Improved method for the refolding of proteins

proteins.

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PCT/DK94/00054

IMPROVED METHOD FOR THE REFOLDING OF PROTEINS

FIELD OF THE INVENTION

This invention relates to recombinant DNA technology and, in particular to protein engineering technologies for the production of correctly folded proteins by expression of genes or gene fragments in a host organism,· heterologous or homologous, as recombinant protein products, by describing novel general principles and methodology for efficient in vitro refolding of misfolded and/or insoluble proteins, including proteins containing disulphide bonds. This invention further relates to the refolding of unfolded or misfolded polypeptides of any other origin. The invention also relates to novel designs of encrypted recognition sites for factor X_a cleavage of chimeric proteins, sites that only become recognized after in vitro derivatization. Two analogues of bovine coagulation factor X_a, suitable for small-, medium-, or large-scale technological applications involving specific cleavage of chimeric proteins at sites designed for cleavage by factor X_a are provided, too. Finally the invention relates to designs of reversible disulphide-blocking reagents, useful as auxiliary compounds for refolding of cysteine-containing proteins, including a general assay procedure by which such disulphide exchange reagents can be evaluated for suitability for this specific purpose.

GENERAL BACKGROUND OF THE INVENTION

Technologies for the production of virtually any polypeptide by introduction, by recombinant DNA methods, of a natural or synthetic DNA fragment coding for this particular polypeptide into a suitable host have been under intense development over the past fifteen years, and are at present essential tools for biochemical research and for a number of industrial processes for production of high-grade protein products for biomedical or other industrial use.

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Four fundamental properties of biological systems render heterologous production of proteins possible:

(i) The functional properties of a protein are entirely specified by its three-dimensional structure, and, due to the molecular environment in the structure, manifested by chemical properties exhibited by specific parts of this structure.

(ii) The three-dimensional structure of a protein is, in turn, specified by the sequence information represented by the specific sequential arrangement of amino acid residues in the linear polypeptide chain(s). The structure information embedded in the amino acid sequence of a polypeptide is by itself sufficient, under proper conditions, to direct the folding process, of which the end product is the completely and correctly folded protein.

(iii) The linear sequence of amino acid residues in the polypeptide chain is specified by the nucleotide sequence in the coding region of the genetic material directing the assembly of the polypeptide chain by the cellular machinery. The translation table governing translation of nucleic acid sequence information into amino acid sequence is known and is almost universal among known organisms and hence allows nucleic acid segments coding for any polypeptide segment to direct assembly of polypeptide product across virtually any cross - species barrier.

(iv) Each type of organism relies on its own characteristic array of genetic elements present within its own genes to interact with the molecular machinery of the cell, which in response to specific intracellular and extracellular factors regulates the expression of a given gene in terms of trans 30 cription and translation.

In order to exploit the protein synthesis machinery of a host cell or organism to achieve substantial production of a desired recombinant protein product, is it therefore neces94/18227

PCT/DK94/00054 sary to present the DNA-segment coding for the desired product to the cell fused to control sequences recognized by the genetic control system of the cell.

The immediate fate of a polypeptide expressed in a host is influenced by the nature of the polypeptide, the nature of the host, and possible host organism stress states invoked during production of a given polypeptide. A gene product expressed in a moderate level and similar or identical to a protein normally present in the host cell, will often undergo normal processing and accumulation in the appropriate cellular compartment or secretion, whichever is the natural fate of this endogenous gene product. In contrast, a recombinant gene product which is foreign to the cell or is produced at high levels often activate cellular defence mechanisms similar to those activated by heat shock or exposure to toxic amino acid analogues, pathways that have been designed by nature to help the cell to get rid of wrong polypeptide material by controlled intracellular proteolysis or by segregation of unwanted polypeptide material into storage particles (inclusion bodies). The recombinant protein in these storage particles is often deposited in a misfolded and aggregated state, in which case it becomes necessary to dissolve the product under denaturing and reducing conditions and then fold the recombinant polypeptide by in vitro methods to obtain a useful protein product.

Expression of eukaryotic genes in eukaryotic cells often allows the direct isolation of the correctly folded and processed gene product from cell culture fluids or from cellular material. This approach is often used to obtain relatively small amounts of a protein for biochemical studies and is presently also exploited industrially for production of a number of biomedical products. However, eukaryotic expression technology is expensive in terms of technological complexity, labour- and material costs. Moreover, the time scale of the development phase required to establish an expression system is at least several months, even for laWO 94/18227

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boratory scale production. The nature and extent of posttranslational modification of the recombinant product often differs from that of the natural product because such modifications are under indirect genetic control in the host cell.

Sequence signals invoking a post-synthetic modification are often mutually recognized among eukaryotes, but availability of the appropriate suit of modification enzymes is given by the nature and state of the host cell.

A variety of strategies have been developed for expression of 10 gene products in prokaryotic hosts, advantageous over eukaryotic hosts in terms of capital, labour and material requirements. Strains of the eubacteria Escherichia coli are often preferred as host cells because £. coli is far better characterized genetically than any other organism, also at the molecular level.

Prokaryotic host cells do not posses the enzymatic machinery required to carry out post-translational modification, and an eukaryotic gene product will therefore necessarily be produced in its unmodified form. Moreover, the product must be synthesized with an N-terminal extension, at least one additional methionine residue arising from the required translation initiation codon, more often also including an N-terminal segment corresponding to that of a highly expressed host protein. General methods to remove such N-terminal extensions by sequence specific proteolysis at linker segments inserted at the junction between the N-terminal extension and the desired polypeptide product have been described (Enterokinase-cleavable linker sequence: EP 035384, The Regents of the University of California; Factor X_a-cleavable linker sequence: EP 161937, Nagai &amp; Thogersen, Assignee: Celltech Ltd.).

Over the years a considerable effort has been directed at the development of strategies for heterologous expression in prokaryotes to generate recombinant protein products in a soluble form or fusion protein constructs that allow secreWO 94/18227 2155335 PCT/DK94/0OO54 tion from the cell in an active, possibly N-terminally processed form, an effort resulting in limited success only, despite recent developments in the chaperone field. Typically, much time and effort is required to develop and modify an expression system before even a small amount of soluble and correctly folded fusion protein product can be isolated. More often all of the polypeptide product is deposited within the host cell in an improperly folded state in inclusion bodies. This is in particular true when expressing eukaryotic proteins containing disulphide bridges.

Available methods for in vitro refolding of proteins all describe processes in which the protein in solution or nonspecifically adsorbed to ion exchange resins etc. is exposed to solvent, the composition of which is gradually changed over time from strongly denaturing (and possibly reducing) to non-denaturing in a single pass. This is often carried out by diluting a concentrated solution of protein containing 6-8 M guanidine hydrochloride or urea into a substantial volume of non-denaturing buffer, or by dialysis of a dilute solution of the protein in the denaturing buffer against the non-denaturing buffer. Numerous variants of this basic procedure have been described, including addition of specific ligands or cofactors of the active protein and incorporation of polymer substances like polyethylene oxide (polyethylene glycol), thought to stabilize the folded structure.

Although efficient variants of the standard in vitro refolding procedure have been found for a number of specific protein products, including proteins containing one or more disulphide bonds, refolding yields are more often poor, and scale-up is impractical and expensive due to the low solubility of most incompletely folded proteins which implies the use of excessive volumes of solvent.

The common characteristic of all traditional in vitro refolding protocols is that refolding induced by sudden or gradual reduction of dénaturant is carried out as a singleWO 94/18227

PCT/DK94/00054 pass operation, the yield of which is then regarded as the best obtainable for the protein in question.

The general field of protein folding has been summarized in a recent text book edited by Thomas W. Creighton (Protein folding, ed. Creighton T.E., Freeman 1992) and a more specific review of practical methods for protein refolding was published in 1989 by Rainer Jaenicke &amp; Rainer Rudolph (p. 191-223 in, Protein Structure, a practical approach, ed. T. E. Creighton, IRL Press 1989). Among the numerous more detailed publications, state-of-the-art reviews like those by Schein (Schein C. H., 1990, Bio/Technology 8, 308-317) or Buchner and Rudolph (Buchner J. and Rudolph R, 1991 Bio/Technology 9, 157-162) may be consulted.

In conclusion, there is a definite need for generally appli15 cable high-yield methods for the refolding of un- or misfolded proteins derived from various sources, such as prokaryotic expression systems or peptide synthesis.

SUMMARY OF THE INVENTION

It has been found by the inventors that refolding yields can be greatly increased by taking into account that the protein folding process is a kinetically controlled process and that interconversion between folded, unfolded and misfolded conformers of the protein are subject to hysteresis and timedependent phenomena that can be exploited to design a cyclic dénaturation-renaturation process, in which refolded protein product accumulates incrementally in each cycle at the expense of unfolded and misfolded conformers, to generate a new refolding process of much greater potential than the basic traditional approach.

By the term folded protein is meant a polypeptide in (a) conformational state(s) corresponding to that or those occurring in the protein in its biologically active form or unique stable intermediates that in subsequent steps may be conWO 94/18227

PCT/DK94/00054 verted to generate the biologically active species. The covalent structure of the folded protein in terms of crosslinking between pairs of cysteine residues in the polypeptide is identical to that of the protein in its bio5 logically active form.

Accordingly, the term unfolded protein refers to a polypeptide in conformational states less compact and welldefined than that or those corresponding to the protein in its biologically active, hence folded, form. The covalent structure of the unfolded protein in terms of crosslinking between pairs of cysteine residues in the polypeptide may or may not be identical to that of the protein in its biologically active form. Closely related to an unfolded protein is a misfolded protein which is a polypeptide in a conformât!15 onal state which is virtually thermodynamically stable, sometimes even more so than that or those states corresponding to the protein in its folded form, but which does not exhibit the same degree, if any, of the biological activity of the folded protein. As is the case for the unfolded pro20 tein, the covalent structure in terms of crosslinking between pairs of cysteine residues in the polypeptide may or may not be the same as that of the folded protein.

By the term refolded protein is meant a polypeptide which has been converted from an unfolded state to attain its biologically active conformation and covalent structure in terms of crosslinking between correct pairs of cysteine residues in the polypeptide.

The new generally applicable protein refolding strategy has been designed on the basis of the following general proper30 ties of protein structure.

(a) The low solubility of unfolded proteins exposed to nondenaturing solvents reflects a major driving force inducing the polypeptide either to form the compact correctly refolded structure or to misfold and generate dead-end aggregates or

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PCT/DK94/00054 precipitates, which are unable to refold and generate the correctly refolded structure under non-denaturing conditions within a reasonable amount of time.

(b) A newly formed dead-end aggregate is more easily dena5 tured i.e. converted into an unfolded form than the correctly refolded protein because the structure of the dead-end aggregate is more disordered. Probably misfolding is also in general a kinetically controlled process.

(c) An unfolded protein is often not (or only very slowly) able to refold into the correctly refolded form at dénaturant levels required to denature dead-end aggregates within a reasonable amount of time.

(d) The body of evidence available to support (b) includes detailed studies of folding and unfolding pathways and inter15 mediates for several model proteins. Also illustrative is the observation made for many disulphide bonded proteins that the stability of disulphide bonds against reduction at limiting concentrations of reducing and denaturing agents is often significantly different for each disulphide bridge of a given protein, and that the disulphide bridges in the folded protein are in general much less prone to reduction or disulphide exchange than non-native disulphide bonds in a denatured protein or protein aggregate.

The new strategy for a refolding procedure is most easily illustrated by way of the following theoretical example:

Consider a hypothetical protein - stably folded in a nondenaturing buffer A and stably unfolded in the strongly denaturing buffer B” (being e.g. a buffer containing 6 M guanidine-HC1) - exposed to buffer A or to buffer B and then subjected to incubation at intermediate levels of dénaturation in mixtures of buffers A and B.

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Levels between e.g. 100-75% B lead to conversion of both folded protein and dead-end aggregated protein to the unfolded form within a short period of time.

Levels between e.g. 75-50% B lead to conversion of newly 5 formed dead-end aggregate to the unfolded form, whereas almost all refolded protein remains in a native-like structure, stable at least within a period of time of hours, from which it may snap back into the refolded form upon removal of the dénaturant.

Levels in excess of 10%B prevent rapid formation of refolded form from unfolded form.

A solvent composition step from 100%B to 0%B converts unfolded protein to dead-end aggregate (75% yield) and refolded protein (25% yield).

Let us now subject a sample of this protein, initially in its unfolded form in 100%B, to a time-series of programmed dénaturation-renaturation cycles as illustrated in Fig. 1, each consisting of a renaturation phase (F_n) (&lt;10%B) and a dénaturation phase (D_n). At the end of the renaturation phase of cycle(i) the dénaturant content is changed to a level, ki % less than the dénaturant level of the previous cycle. Following a brief incubation the dénaturant is again removed, and the next renaturation phase F_i+1 entered. Assuming the dénaturation level starts out at 100%B and ki for each cycle is fixed at 4%, this recipe will generate a damped series of dénaturation steps dying out after 25 cycles.

Through 25 cycles, as outlined above, the accumulation of refolded protein would progress as follows:

In cycles 1 to 5 all of the protein, folded as well as misfolded will become unfolded in each of the dénaturation phases D_n.

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Cycles 7 through 12: Dead-end aggregates will be converted to unfolded protein in each step whereas protein recoverable as refolded product will accumulate in the following amounts, cycle by cycle: 25%, 44%, 58%, 68%,

76% and 82%.

No further conversions take place through cycles 13 to 25.

The cyclic refolding process would therefore produce a total refolding yield of over 80%, whereas traditional one-pass renaturation at best would produce a yield of 25%.

It will be appreciated that a great number of simplifying approximations in terms of all-or-none graduation of each characteristic of the various conformational states of the hypothetical protein have been made. The basic working prin15 ciple, nevertheless, remains similar if a more complicated set of presumptions are incorporated in the model.

Arranging a practical setup for establishing a cyclic denaturation/renaturation protein refolding process can be envisaged in many ways.

The protein in solution could e.g. be held in an ultrafiltration device, held in a dialysis device or be confined to one of the phases of a suitable aqueous two-phase system, all of which might allow the concentration of low-molecular weight chemical solutes in the protein solution to be con25 trolled by suitable devices.

Alternatively, the protein could be adsorbed to a suitable surface in contact with a liquid phase, the chemical composition of which could be controlled as required. A suitable surface could e.g. be a filtration device, a hollow-fibre device or a beaded chromatographic medium. Adsorption of the protein to the surface could be mediated by non-specific interactions, e.g. as described in WO 86/05809 (Thomas Edwin

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Creighton), by folding-compatible covalent bonds between surface and protein or via specific designs of affinity handles in a recombinant derivative of the protein exhibiting a specific and dénaturation-resistant affinity for a suitably derivatized surface.

The specific implementation of the cyclic denaturation/renaturation protein refolding process established to investigate the potential of the general method was based on a design of cleavable hybrid proteins (EP 161937, Nagai &amp;

Thogersen, Assignee: Celltech Ltd.) containing a metal affinity handle module (EP 0282042 (Heinz Dôbeli, Bernhard Eggimann, Reiner Gentz, Erich Hochuli; Hoffmann-La Roche)) inserted N-terminally to the designed factor X_a cleavage site. Recombinant proteins of this general design, adsorbed on Nickel-chelating agarose beads could then be subjected to the present cyclic refolding process in a chromatographic column refolding reactor perfused with a mixture of suitable denaturing and non-denaturing buffers, delivered by an array of calibrated pumps, the flow rates of which was time20 programmed through computer control.

A general scheme of solid-state refolding entails cycling the immobilized protein as outlined above or by any other means and implementations between denaturing and non-denaturing conditions in a progressive manner, in which the concentra25 tion of the denaturing agent is gradually reduced from high starting values towards zero over a train of many renaturation-denaturation cycles. Using this approach it is not necessary to determine precisely which limiting dénaturant concentration is required to obtain folding yield enrichment in the course of cycling of the specific protein at hand, because the progressive train of cycles will go through (up to) three phases, an early phase in which folded product present at the end of cycle (i) is completely denatured at the dénaturation step of cycle (i+1), an intermediate produc35 tive phase during which refolded protein accumulates in increasing quantity, and a late phase during which the conWO 94/18227

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centration of dénaturant is too low to perturb the refolded protein or any remaining misfolded structures. Subjecting the protein to a progressing series of dénaturation-renaturation cycles as outlined will therefore include several productive cycles.

For disulphide-containing proteins progressive dénaturationrenaturation cycling may be enhanced by using equipment similar to advanced chromatography equipment with on-line facilities to monitor buffer compositions of folding reactor effluent. Information on effluent composition with regard to reductant and disulphide reshuffling reagent concentration profile would reveal productive cycling, and could therefore be used as input to an intelligent processor unit, in turn regulating the progression of dénaturant concentration in a feed-back loop to ensure that most of the cycling effort is spent within the productive phase of the dénaturation-renaturation cycle train. Such auto-optimization of cycling conditions would be possible because the analytical system may be used to measure extent and direction of changes in redox equilibrium in the buffer stream, measurements that directly reflect titration of thiol-groups /disulphide equivalents in the immobilized protein sample, and is therefore directly translatable into average number of disulphide bonds being disrupted or formed during the various phases of a cycle.

Other possible inputs for the intelligent processor controlling the progression of cycling include measurements of ligand-binding, substrate conversion, antibody binding ,ability and, indeed, any other interacting soluble agent interacting in distinct ways with misfolded and folded protein, which in the assessing stage of folding measurement might be percolated through the refolding reactor and then in-line monitored in the effluent by suitable analytical devices.

An intelligent monitoring and control system could furthermore use the available information to direct usable portions

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PCT/DK94/00054 of reactor effluent to salvage/recycling subsystems thereby minimizing expenses for large scale operations.

After execution of the folding procedure the final product may be eluted from the affinity matrix in a concentrated form, processed to liberate the mature authentic protein by cleavage at the designed protease cleavage site and then subjected to final work-up using standard protein purification and handling techniques, well-known within the field of protein chemistry.

DETAILED DISCLOSURE OF THE INVENTION

Thus, the present invention relates to a method for generating a processed ensemble of polypeptide molecules, in which processed ensemble the conformational states represented contain a substantial fraction of polypeptide molecules in one particular uniform conformation, from an initial ensemble of polypeptide molecules which have the same amino acid sequence as the processed ensemble of polypeptide molecules, comprising subjecting the initial ensemble of polypeptide molecules to a series of at least two successive cycles each of which comprises a sequence of

1) at least one denaturing step involving conditions exerting a denaturing influence on the polypeptide molecules of the ensemble followed by

2) at least one renaturing step involving conditions having a renaturing influence on the polypeptide molecules having conformations resulting from the preceding step.

In the present specification and claims, the term ensemble is used in the meaning it has acquired in the art, that is, it designates a collection of molecules having essential common features. Initially (an initial ensemble), they have at least their amino acid sequence in common (and of course retain this common feature). When the ensemble of polypeptide

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2135335 molecules has been treated in the method of the invention (to result in a processed ensemble), the conformational states represented in the ensemble will contain a substantial fraction of polypeptide molecules with one particular conforma5 tion. As will be understood from the discussion which follows, the substantial fraction of polypeptide molecules with one particular conformation in the processed ensemble may vary dependent on the parameters of the treatment by the method of 10 the invention, the size of the protein in the particular conformation, the length and identity of the amino acid sequence of the molecules, etc. In the examples reported herein, in which the process parameters have not yet been optimized, the fraction of polypeptide molecules with one particular conformation varied between 15% and 100% of the ensemble, which in all cases is above what could be obtained prior to the present invention. In example 13 it is further demonstrated that purification of the polypeptide molecules prior to their subjection to the method of the invention increases the fraction of polypeptide molecules with one particular conformation.

Denaturing step refers to exposure of an ensemble of polypeptide molecules during a time interval to physical and/or chemical circumstances which subject the ensemble of polypeptide molecules to conditions characterized by more severe denaturing power than those characterizing conditions immediately prior to the denaturing step.

Accordingly, the term renaturing step refers to exposure of an ensemble of polypeptide molecules during a time interval to physical and/or chemical circumstances which subject the ensemble of polypeptide molecules to conditions characterized by less severe denaturing power than those characterizing conditions immediately prior to the denaturing step.

It will be understood, that the substantial fraction men35 tioned above will depend in magnitude on the ensemble of

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PCT/DK94/00054 polypeptide molecules which are subjected to the method of the invention. If the processed ensemble of polypeptides consists of monomeric proteins of relatively short lengths and without intramolecular disulphide bridges the method will in general result in very high yields, whereas complicated molecules (such as polymeric proteins with a complicated disulphide bridging topology) may result in lower yields, even if the conditions of the method of the invention are fully optimized.

An interesting aspect of the invention relates to a method described above wherein the processed ensemble comprises a substantial fraction of polypeptide molecules in one conformational state the substantial fraction constituting at least 1% (w/w) of the initial ensemble of polypeptide mo15 lecules. Higher yields are preferred, such as at least 5%, at least 10%, at least 20%, and at least 25% of the initial ensemble of polypeptide molecules. More preferred are yields of at least 30%, such as at least 40%, 50%, 60%, 70%, and at leat 80%. Especially preferred are yields of at least 85%, such as 90%, 95%, 97%, and even at 99%. Sometimes yields close to 100% are observed.

When the polypeptide molecules of the ensemble contain cysteine, the processed ensemble will comprise a substantial fraction of polypeptide molecules in one particular uniform conformation which in addition have substantially identical disulphide bridging topology.

In most cases, the polypeptide molecules subjected to the method of the invention will be molecules which have an amino acid sequence identical to that of an authentic polypeptide, or molecules which comprise an amino acid sequence corresponding to that of an authentic polypeptide joined to one or two additional polypeptide segments.

By the term authentic protein or polypeptide is meant a polypeptide with primary structure, including N- and C-terWO 94/18227 ?·

PCT/DK94/00054 minai structures, identical to that of the corresponding natural protein. The term also denotes a polypeptide which has a known primary structure which is not necessarily identical to that of a natural protein, which polypeptide is the intentional end-product of a protein synthesis.

By the term natural protein” is meant a protein as isolated in biologically active form from an organism, in which it is present not as a consequence of genetic manipulation.

In contrast, the term artificial protein or polypeptide as used in the present specification and claims is intended to relate to a protein/polypeptide which is not available from any natural sources, i.e. it cannot be isolated and purified from any natural source. An artificial protein/polypeptide is thus the result of human intervention, and may for instance be a product of recombinant DNA manipulation or a form of in vitro peptide synthesis. According to the above definitions such an artificial protein may be an authentic protein, but not a natural protein.

Thus, the invention also relates to a method wherein natural proteins as well as artificial proteins are subjected to the refolding processes described herein.

As will be explained in greater detail below, it may be advantageous for various reasons that the authentic polypeptide is joined to polypeptide segments having auxil25 iary functions during the cycling and other previous or subsequent processing, e.g. as handles for binding the polypeptide to a carrier, as solubility modifiers, as expression boosters which have exerted their beneficial function during translation of messenger RNA, etc. Such an auxiliary polypeptide segment will preferably be linked to the authentic polypeptide via a cleavable junction, and where two such auxiliary polypeptide segments are linked to the authentic polypeptide, this may be via similar cleavable junctions which will normally be cleaved simultaneously, or through

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PCT/DK94/00054 dissimilar cleavable junctions which may be cleaved in any time sequence.

In accordance with what is explained above, it is believed to be a major novel characteristic feature of the present inven5 tion that the cycling (which, as explained above, comprises at least two successive cycles) will give rise to at least one event where a renaturing step is succeeded by a denaturing step where at least a substantial fraction of the refolded polypeptides will be denatured again.

In most cases, the processing will comprise at least 3 cycles, often at least 5 cycles and more often at least 8 cycles, such as at least 10 cycles and, in some cases at least 25 cycles. On the other hand, the series of cycles will normally not exceed 2000 cycles and will often comprise at most 1000 cycles and more often at most 500 cycles. The number of cycles used will depend partly on the possibilities made available by the equipment in which the cycling is performed.

Thus, if the cycling treatment is performed with the poly20 peptide molecules immobilized to a carrier column, such as will be explained in greater detail below, the rate with which the liquid phase in contact with the column can be exchanged will constitute one limit to what can realistically be achieved. On the other hand, high performance liquid chromatography (HPLC) equipment will permit very fast exchange of the liquid environment and thus make cycle numbers in the range of hundreds or thousands realistic.

Other consideration determining the desirable number of cycles are, e.g., inherent kinetic parameters such as inter30 conversion between cis and trans isomers at proline residues which will tend to complicate redistribution over the partially folded states and will thus normally require due consideration of timing. Another time-critical characteristic

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PCT/DK94/00054 resides m the kinetics of disulphide reshuffling (cf. the discussion below of disulphide-reshuffling systems).

With due consideration of the above, the cycling series will often comprise at most 200 cycles, more often at most 100 cycles and yet more often at most 50 cycles.

In accordance with what is stated above, the duration of each denaturing step may be a duration which, under the particular conditions in question, is at least one millisecond and at most one hour, and the duration of each renaturing step may be a duration which, under the particular conditions in question, is at least 1 second and at most 12 hours.

In most embodiments of the method, the denaturing conditions of each individual denaturing step are kept substantially constant for a period of time, and the renaturing conditions of each individual renaturing step are kept substantially constant for a period of time, the periods of time during which conditions are kept substantially constant being separated by transition periods during which the conditions are changed. The transition period between steps for which condi20 tions are kept substantially constant may have a duration varying over a broad range, such as between 0.1 second and 12 hours and will normally be closely adapted to the durations of the denaturing and renaturing steps proper.

Bearing this in mind, the period of time for which the dena25 turing conditions of a denaturing step are kept substantially constant may, e.g. have a duration of at least one millisecond and at most one hour, often at most 30 minutes, and the period of time for which the renaturing conditions of a renaturing step are kept substantially constant has a du30 ration of at least 1 second and at most 12 hours, and often at most 2 hours.

In practice, the period of time for which the denaturing conditions of a denaturing step are kept substantially con94/18227

PCT/DK94/00054 stant will often have a duration of between 1 and 10 minutes, and the period of time for which the renaturing conditions of a renaturing step are kept substantially constant will often have a duration of between 1 and 45 minutes.

It will be understood from the above, that adjustments should be made to the intervals stated above, taking into consideration the change of kinetics resulting from the change in physical conditions to which the polypeptides are subjected. For instance, the pressure may be very high (up to 5000 Bar) when using an HPLC system when performing the method of the invention, and under such circumstances very rapid steps may be accomplished and/or necessary. Further, as can be seen from the examples, the temperature parameter is of importance, as some proteins only will refold properly at temperatures far from the physiological range. Both temperature and pressure will of course have an effect on the kinetics of the refolding procedure of the invention, and therefore the above-indicated time intervals of renaturing and denaturing steps are realistic boundaries for the many possible embodiments of the invention.

For a given utilization of the method of the invention, the skilled person will be able to determine suitable conditions based, e.g., on preliminary experiments.

As indicated above, the polypeptide molecules are normally in contact with a liquid phase during the denaturing and renaturing steps, the liquid phase normally being an aqueous phase. This means that any reagents or auxiliary substances used in the method will normally be dissolved in the liquid phase, normally in an aqueous phase. However, if convenient, the liquid phase may also be constituted by one or more organic solvents .

In connection with renaturing of proteins, it is well known to use a so-called chaperone or chaperone complex. Chaperones are a group of recently described proteins that show a

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PCT/DK94/00054 common feature in their capability of enhancing refolding of unfolded or partly unfolded proteins. Often, the chaperones are .multimolecular complexes. Many of these chaperones are heat-shock proteins, which means that in vivo, they are serving as factors doing post-traumatic repair on proteins that have been destabilized by the trauma. To be able to fulfil this function, chaperones tend to be more stable to traumatic events than many other proteins and protein complexes. While the method of the invention does not depend on the use of a molecular chaperone or a molecular chaperone complex, it is, of course, possible to have a suitable molecular chaperone or molecular chaperone complex present during at least one renaturing step, and it may be preferred to have a molecular chaperone or a molecular chaperone complex present during substantially all cycles.

As mentioned above, the polypeptide molecules are preferably substantially confined to an environment which allows changing or exchanging the liquid phase substantially without entraining the polypeptide molecules.

This can be achieved in a number of ways. For instance, the polypeptide molecules may be contained in a dialysis device, or they may be confined to one of the phases of a suitable liquid two-phase system. Such a suitable aqueous two phase system may, e.g., contain a polymer selected from the group consisting of polyethylene oxide (polyethylene glycol), polyvinyl acetate, dextran and dextran sulphate. In one interesting setup, one phase contains polyethylene oxide (polyethylene glycol) and the other phase contain dextran, whereby the polypeptide molecules will be confined to the dextran-containing phase.

Another way of avoiding entraining the polypeptide by having the polypeptide molecules bound to a solid or semisolid carrier, such as a filter surface, a hollow fibre or a beaded chromatographic medium, e.g. an agarose or polyacrylamide gel, a fibrous cellulose matrix or an HPLC or FPLC (Fast Performance Liquid Chromatography) matrix. As another

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PCT/DK94/00054 measure, the carrier may be a substance having molecules of such a size that the molecules with the polypeptide molecules bound thereto, when dissolved or dispersed in a liquid phase, can be retained by means of a filter, or the carrier may be a substance capable of forming micelles or participating in the formation of micelles allowing the liquid phase to be changed or exchanged substantially without entraining the micelles.

In cases where the micelle-forming components would tend to escape from the system as monomers, e.g. where they would be able to some extent to pass an ultrafilter used in confining the system, this could be compensated for by replenishment will additional micelle-forming monomer.

The carrier may also be a water-soluble polymer having molecules of a size which will substantially not be able to pass through the pores a filter or other means used in confining the system.

The polypeptide molecules are suitably non-covalently adsorbed to the carrier through a moiety having affinity to a component of the carrier. Such a moiety may, e.g., be a

0 biotin group or an analogue thereof bound to an amino acid moiety of the polypeptide, the carrier having avidin, streptavidin or analogues thereof attached thereto so as to establish a system with a strong affinity between the thus modified polypeptide molecules and the thus modified carrier.

It will be understood that he affinity between the modified polypeptide and the modified carrier should be sufficiently stable so that the adsorption will be substantially unaffected by the denaturing conditions; the removal of the polypeptide molecules from the carrier after the cycling should be performed using specific cleaving, such as is explained in the following.

An example of a suitable amino acid residue to which a biotinyl group may be bound is lysine.

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One interesting way of introducing an amino acid carrying a moiety having affinity to the carrier is CPY synthesis. CPY (carboxy peptidase Y) is known to be capable of adding amino acid amide irrespective of the nature of the side chain of that amino acid amide.

In an interesting embodiment, the moiety having affinity to the carrier is the polypeptide segment SEQ ID NO: 47, in which case the carrier suitably comprises a Nitrilotriacetic Acid derivative (NTA) charged with Ni⁺⁺ ions, for instance an

NTA-agarose matrix which has been bathed in a solution comprising Ni⁺⁺.

An important aspect of the invention relates to the presence of suitable means in the polypeptide molecule preparing the molecule for later cleavage into two or more segments, where15 in one segment is an authentic polypeptide as defined above. Such combined polypeptide molecules (fusion polypeptide molecules) may for this purpose comprise a polypeptide segment which is capable of directing preferential cleavage by a cleaving agent at a specific peptide bond. The polypeptide segment in question may be one which directs the cleavage as a result of the conformation of the segment which serves as a recognition site for the cleaving agent.

The cleavage-directing polypeptide segment may for instance be capable of directing preferential cleavage at a specific peptide bond by a cleaving agent selected from the group consisting of cyanogen bromide, hydroxylamine, iodosobenzoic acid and N-bromosuccinimide.

The cleavage-directing polypeptide segment may be one which is capable of directing preferential cleavage at a specific peptide bond by a cleaving agent which is an enzyme and one such possible enzyme is bovine enterokinase or an analogue and/or homologue thereof.

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In an important aspect of the invention, the cleaving agent is the enzyme bovine coagulation factor X_a or an analogue and/or homologue thereof (such analogues will be discussed in greater detail further below), and the polypeptide segment which directs preferential cleavage is a sequence which is substantially selectively recognized by the bovine coagulation factor X_a or an analogue and/or homologue thereof. Important such segments are polypeptide segments that have a sequence selected from the group consisting of SEQ ID NO: 38,

SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

An interesting feature of the invention is the possibility of masking and unmasking polypeptide segments with respect to their ability to direct cleavage at a specific peptide bond, whereby it is obtained that different segments of the polypeptide can be cleaved at different stages in the cycles.

Thus, when the polypeptide molecules comprise a polypeptide segment which is in vitro-convertible into a derivatized polypeptide segment capable of directing preferential cleavage by a cleaving agent at a specific peptide bond, a mas20 king/unmasking effect as mentioned becomes available. An especially interesting version of this strategy is where the in vitro-convertible polypeptide segment is convertible into a derivatized polypeptide segment which is substantially selectively recognized by the bovine coagulation factor X_a or an analogue and/or homologue thereof.

It is contemplated that both cysteine and methionine residues can be converted into modified residues, which modified residues make the segments having amino acid sequences selected from the group consisting of SEQ ID NO: 43, SEQ ID

NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 in vitro-convertible into segments recognized by bovine coagulation factor X_a or an analogue and/or homologue thereof.

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According to the invention, one possible solution involving the cysteine residue is that a polypeptide segment with the amino acid sequence SEQ ID NO: 43 or SEQ ID NO: 44, is converted into a derivatized polypeptide which is substantially selectively recognized by bovine coagulation factor X_a, by reacting the cysteine residue with N-(2-mercaptoethyl)morpholyl-2-thiopyridyl disulphide or mercaptothioacetate-2-thiopyridyl disulphide.

A possible strategy according to the invention involving 10 methionine is that a polypeptide segment with the amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 46, is converted into a derivatized polypeptide, which is substantially selectively recognized by bovine coagulation factor X_a, by oxidation of the thioether moiety in the methionine side group to a sulph15 oxide or sulphone derivative.

Preferred embodiments of the method according to the invention are those wherein the cleavage-directing segments with the amino acid sequences SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42, or the masked cleavage-directing segments with the amino acid sequences SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 are linked N-terminally to the authentic polypeptide, because then no further processing other than the selective cleaving is necessary in order to obtain the authentic polypeptide in solution. On the other hand, one possible reason for linking the cleavage directing sequences at the C-terminal end of the authentic polypeptide would be that the correct folding of the polypeptide molecules is dependent on a free N-terminal of the polypeptide molecules. In such a case, the part of the cleaving-directing sequence remaining after cleaving can be removed by suitable use of carboxypeptidases A and B.

The change of conditions during the transition period between the steps may according to the invention be accomplished by changing the chemical composition of the liquid phase with which the polypeptide molecules are in contact. Thus, denaPCT/DK94/00054

WO 94/18227 turing of the polypeptide molecules may be accomplished by contacting the polypeptide molecules with a liquid phase in which at least one denaturing compound is dissolved, and renaturing of the polypeptide molecules is accomplished by contacting the polypeptide molecules with a liquid phase which either contains at least one dissolved denaturing compound in such a concentration that the contact with the liquid phase will tend to renature rather than denature the ensemble of polypeptide molecules in their respective confor10 mation states resulting from the preceding step, or contains substantially no denaturing compound.

The expression denaturing compound refers to a compound which when present as one of the solutes in a liquid phase comprising polypeptide molecules may destabilize folded states of the polypeptide molecules leading to partial or complete unfolding of the polypeptide chains. The denaturing effect exerted by a denaturing compound increases with increasing concentration of the denaturing compound in the solution, but may furthermore be enhanced or moderated due to the presence of other solutes in the solution, or by changes in physical parameters, e.g. temperature or pressure.

As examples of suitable denaturing compounds to be used in the method according to the invention may be mentioned urea, guanidine - HC1, di-C_1-6alkylf ormamides such as dimethylform25 amide and di - - alkylsulphones.

The liquid phase used in at least one of the denaturing steps and/or in at least one of the renaturing steps may according to the invention contain a least one disulphide-reshuffling system.

Disulphide reshuffling systems are redox systems which contain mixtures of reducing and oxidating agents, the presence of which facilitate the breaking and making of disulphide bonds in a polypeptide or between polypeptides. Accordingly, disulphide reshuffling agents or disulphide

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PCT/DK94/00054 reshuffling compounds are such reducing and oxidating agents which facilitate the breaking and making of disulphide bonds in a polypeptide or between polypeptides. In an important aspect of the invention, the disulphide-reshuffling system contained in the aqueous phase which is in contact with the proteins comprises as a disulphide reshuffling system a mixture of a mercaptan and its corresponding disulphide compound.

As an example, all cysteine residues in the polypeptide 10 molecules may have been converted to mixed disulphide products of either glutathione, thiocholine, mercaptoethanol or mercaptoacetic acid, during at least one of the denaturing/renaturing cycles. Such a converted polypeptide is termed a fully disulphide-blocked polypeptide or protein and this term thus refers to a polypeptide or a protein in which cysteine residues have been converted to a mixed-disulphide in which each cysteine residue is disulphide-linked to a mercaptan, e.g. glutathione. The conversion of the cysteine residues to mixed disulphide products may be accomplished by reacting a fully denatured and fully reduced ensemble of polypeptide molecules with an excess of a reagent which is a high-energy mixed disulphide compounds, such as aliphaticaromatic disulphide compounds, e.g. 2-thiopyridyl glutathionyl disulphide, or by any other suitable method.

As examples of high-energy mixed disulphides, that is, mixed disulphides having a relatively unstable S-S bond) may be mentioned mixed disulphides having the general formula:

*2

I

R₁-S-S-C-R₃

I r₄ wherein R_x is 2-pyridyl, and each of R₂, R₃ and R₄ is hydrogen or an optionally substituted lower aromatic or aliphatic

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<img file="CA2155335C_D0006.tif" />

PCT/DK94/00054 hydrocarbon group. Examples of such mixed disulphides are glutathionyl- 2 - thiopyridyl disulphide, 2 -thiocholy1-2 -thiopyridyl disulphide, 2 -mercaptoethanol- 2 -thiopyridyl disulphide and mercaptoacetate-2 - thiopyridyl disulphide.

In interesting embodiments, the disulphide-reshuffling system contains glutathione, 2-mercaptoethanol or thiocholine, each of which in admixture with its corresponding symmetrical disulphide.

The suitability of a given mixture of thiols for use as selective reducing and/or disulphide-reshuffling system in a cyclic refolding/reoxidation procedure for a specific protein product can be directly assayed by incubating ensembles of samples of a mixture of folded and misfolded protein with an array of thiol mixtures at several different concentrations of dénaturant exerting weakly, intermediate or strongly denaturing effects on the protein. Following incubation, the disulphide topology in each sample is then locked by reaction with an excess of thiol-blocking reagent (e.g. Iodoacetamide) before subjecting each set of samples to SDS-PAGE under non20 reducing conditions. Correctly disulphide-bridged material and material in undesired covalent topological states will appear in separate bands and will therefore allow quantitative assessment of folding state of the protein at the time of thiol-blocking, because only correctly unique disulphide25 bonded topoisomer may correspond to correctly folded protein present at the end of incubation with thiol/disulphide and dénaturant agents. This set of experiments allows identification of the range of dénaturant levels at which a given thiol/disulphide reagent may be advantageously used as disulphide reshuffling agent, as revealed by preferential reduction and reshuffling of wrong disulphide bonds and low tendency to reduce bonds in the fully folded protein. This reagent testing procedure may be used as a general procedure for selecting advantageous reducing and/or thiol/disulphide reshuffling reagents. Example 12 demonstrates application of this analytical procedure to assess the suitability for

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PCT/DK94/00054 selective reduction of misfolded forms of a model protein for 5 thiol reagents and thereby demonstrates the operability of the above procedure.

It will be understood that the above-indicated procedure for 5 selecting suitable disulphide reshuffling systems may also be employed for selecting other compositions than mixtures of thiols. Any mixture containing suitable reducing/oxidating agents may be evaluated according to the above indicated procedure, and the composition of choice in the method of the invention will be the one which shows the highest ability of preferentially reduce incorrectly formed disulphide bridges.

Thus, a very important aspect of the invention is a method for protein refolding as described herein, wherein at least one disulphide-reshuffling system contained in liquid phase in at least one renaturing and/or denaturing step is one which is capable of reducing and/or reshuffling incorrectly formed disulphide bridges under conditions with respect to concentration of the denaturing agent at which unfolded and/or misfolded proteins are denatured and at which there is substantially no reduction and/or reshuffling of correctly formed disulphide bridges.

An interesting embodiment of the invention is a method as described above, wherein a disulphide reshuffling system is used in at least one denaturing/renaturing step and resulting in a ratio between the relative amount of reduced/reshuffled initially incorrectly formed disulphide bridges and the relative amount of reduced/reshuffled initially correctly formed disulphide bridges of at least 1.05. The ratio will preferably be higher, such as 1.1, 1.5, 2.0, 3.0, 5.0, 10,

100, 1000, but even higher ratios are realistic and are thus especially preferred according to the invention.

By the terms initially incorrectly/correctly with respect to the form of disulphide bridges is meant the disulphide

WO 94/18227 2155335 PCT/DK94/00054 bridging topology just before the disulphide reshuffling system exerts its effects.

It will be understood that the ratio has to be greater than 1 in order to allow the net formation of correctly formed disulphide bridges in a protein sample. Normally the ratio should be as high as possible, but even ratios which are marginally above 1 will allow the net formation of correctly formed disulphide bridges in the method of the invention, the important parameter in ensuring a high yield being the number of denaturing/renaturing cycles. Ratios just above one require that many cycles are completed before a substantive yield of correctly formed disulphide bridges is achieved, whereas high ratios only require a limited number of cycles.

In cases where only one disulphide reshuffling system is going to be employed such a disulphide reshuffling system may according to the invention be selected by

1) incubating samples of folded and misfolded protein of the same amino acid sequence as the protein to be processed in the method of the invention with an array of disulphide reshuffling systems at several different concentrations of a chosen denaturing agent,

2) assessing at each of the different concentrations of denaturing agent the ability of each of the disulphide reshuffling systems to reduce and/or reshuffle initially incorrectly formed disulphide bridges without substantially reducing and/or reshuffling initially correctly formed disulphide bridges as assessed by calculating the ratio between the relative amount of reduced/reshuffled initially incorrectly formed disulphide bridges and the relative amount of reduced/reshuffled initially correctly formed disulphide bridges, and

3) selecting as the disulphide reshuffling system X, the disulphide reshuffling system which exhibit the capa30

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PCT/DK94/00054 bility of reducing initially incorrectly formed disulphide bridges without substantially reducing and/or reshuffling initially correctly formed disulphide bridges in the widest range of concentrations of the chosen denaturing agent.

Alternatively more than one disulphide reshuffling system may be employed, for instance in different cycles in the cyclic refolding method of the invention, but also simultaneously in the same cycles. This will e.g. be the case when it is likely or has been established by e.g. the method outlined above that the overall yield of correctly folded protein with correct disulphide bridging topology will be higher if using different disulphide reshuffling systems in the method of the invention.

In order to calculate the above-indicated the ratio between the relative amount of reduced/reshuffled initially incorrectly formed disulphide bridges and the relative amount of reduced/reshuffled initially correctly formed disulphide bridges, the following method may be employed: to the initial mixture of reactants in step 1) is added a known amount of radioactively labelled correctly folded protein. When the amounts of correctly and incorrectly folded protein are assessed in step 2) (for instance by non-reducing SDS-PAGE) the content of radioactivity in the correctly folded protein fraction is determined as well. Thereby an assessment of the now incorrectly folded (but initially correctly folded) protein can be determined in parallel with the determination of the total distribution of correctly/incorrectly folded protein. The above-mentioned ratio can thus be calculated as

<img file="CA2155335C_D0007.tif" />

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PCT/DK94/00054 wherein and C₂ are the initial and the final amounts of correctly folded proteins, respectively, U_x is the amount of initially incorrectly folded protein, and A_x and A₂ are the radioactivity in the initial correctly folded protein frac5 tion and in the final correctly folded protein, respectively.

In addition to the denaturing means mentioned above, denaturing may also be achieved or enhanced by decreasing pH of the liquid phase, or by increasing pH of the liquid phase.

The polarity of the liquid phase used in the renaturing may according to the invention have been modified by the addition of a salt, a polymer and/or a hydrofluoro compound such as trifluoroethanol.

According to the invention, the denaturing and renaturing of the polypeptide molecules may also be accomplished by direct changes in physical parameters to which the polypeptide molecules are exposed, such as temperature or pressure, or these measures may be utilized to enhance or moderate the denaturing or renaturing resulting from the other measures mentioned above.

However, it will be understood that a most important practical embodiment of the method is performed by accomplishing chemical changes in the liquid phase by changing between a denaturing solution B and a renaturing solution A. In this case, the concentration of one or more denaturing compounds in B will often be adjusted after each cycle, and as one important example, the concentration of one or more denaturing compounds in B will be decremented after each cycle, but in another important embodiment, the concentration of one or more denaturing compounds in medium B is kept constant in each cycle.

This embodiment of the invention, wherein the concentration of denaturing compound(s) medium B is kept constant, is

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PCT/DK94/00054 especially interesting when the most productive phase of the cycling process (with respect to correctly folded protein) has been identified, and large scale production of correctly folded protein is desired. As will be understood, the pre5 ferred concentration(s) of denaturing compound(s) of medium B in this embodiment is the concentration(s) which has been established to ensure maximum productivity in the cyclic process according to the invention.

The polypeptide molecules of the ensemble which is subjected 10 to the method of the invention normally have a length of at least 25 amino acid residues, such as at least 30 amino acid residues or at least 50 amino acid residues.

On the other hand, the polypeptide molecules of the ensemble normally have a length of at most 5000 amino acid residues, such as at most 2000 amino acid residues or at most 1000 or 800 amino acid residues.

As can be seen from example 10, the method of the invention has made possible the production of correctly folded diabody molecules (diabodies are described in Holliger et al., 1993).

An important aspect of the invention therefore relates to a method for producing correctly folded diabody molecules, wherein an initial ensemble of polypeptide molecules comprising unfolded and/or misfolded polypeptides having amino acid sequences identical to the amino acid sequences of monomer fragments of diabody molecules is subjected to a series of at least two successive cycles, each of which comprises a sequence of

1) at least one denaturing step involving conditions exerting a denaturing influence on the polypeptide mole30 cules of the ensemble followed by

2) at least one renaturing step involving conditions having a renaturing influence on the polypeptide mole2155335

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PCTZDK94/00054 cules having conformations resulting from the preceding step, the series of cycles being so adapted that a substantial fraction of the initial ensemble of polypeptide molecules is converted to a fraction of correctly folded diabody molecules.

Such a method for the correct folding of diabodies can be envisaged in any of the above-mentioned scenarios and aspects of the refolding method of the invention, that is, with respect to the choice of physical/chemical conditions as well as cycling schedules. However, an important aspect of the method for correct folding of diabodies is a method as the above-identified, wherein the polypeptide molecules are in contact with a liquid phase containing at least one disulphide reshuffling system in at least one denaturing or renaturing step. The preferred denaturing agent to be used in such a liquid phase is urea, and the preferred disulphide reshuffling system comprises glutathione as the main reducing agent.

A particular aspect of the invention relates to a polypeptide which is a proenzyme of a serine protease, but is different from any naturally occurring serine protease and, in particular, has an amino acid sequence different from that of bovine coagulation factor X (Protein Identification Resource (PIR), National Biomedical Research Foundation, Georgetown University, Medical Center, U.S.A., entry: Pl;EXBO) and which can be proteolytically activated to generate the active serine protease by incubation of a solution of the polypeptide in a non-denaturing buffer with a substance that cleaves the polypeptide to liberate a new N-terminal residue, the substrate specificity of the serine protease being identical to or better than that of bovine blood coagulation factor X_a, as assessed by each of the ratios

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<img file="CA2155335C_D0008.tif" />

(k(I)/k(V) and k(III)/k(V) between cleavage rate against each of the substrates I and III:

I:

III:

Benzoyl-Val-Gly-Arg-paranitroanilide, Tosyl-Gly-Pro-Arg-paranitroanilide, versus that against the substrate

V:

Benzoyl-Ile-Glu-Gly-Arg-paranitroanilide at 20°C, pH=8 in a buffer consisting of 50 mM Tris, 100 mM NaCl, 1 mM CaCl₂, being identical to or lower than the corresponding ratio determined for bovine coagulation factor X_a which is substantially free from contaminating proteases .

The characterization of the above-identified new polypeptides as serine proteases is in accordance with the normal nomenclatural use of the term serine proteases. As is well known in the art, serine proteases are enzymes which are believed to have a catalytic system consisting of an active site serine which is aligned with a histidine residue, and it is believed that the activation of the enzymes from the corresponding proenzymes is based on the liberation of a new N20 terminal residue, the ce-amino group of which is capable of repositioning within the polypeptide structure to form a salt bridge to an aspartic acid residue preceding an active-site serine residue, thereby forming the catalytic site characteristic of serine proteases.

The artificial serine proteases defined above are extremely valuable polypeptide cleaving tools for use in the method of the invention and in other methods where it is decisive to have a cleaving tool which will selectively cleave proteins, even large folded proteins. Analogously to bovine coagulation factor X_a, the above-defined artificial serine proteases in activated form are capable of selectively recognizing the cleaving-directing polypeptide segment SEQ ID NO: 38, but in

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PCT/DK94/00054 contrast to bovine coagulation factor X_a, they can be established with such amino acid sequences that they can be readily produced using recombinant DNA techniques. Thus, the preferred artificial serine proteases of the invention are ones which have amino acid sequences allowing their synthesis by recombinant DNA techniques, in particular in a prokaryote cells such as JB. coli. As will appear from the following discussion and the examples, the artificial serine proteases of the invention, when produced in a prokaryote, may be given an enzymatically active conformation, in which the catalytically active domains are suitably exposed, by cycling according to the method of the present invention.

The quantitative test for selectivity of the artificial serine proteases involves determination of the cleavage rate, k, determined as the initial slope of a curve of absorption of light at 405 nm (absorption maximum of free paranitroaniline) versus time at 20°C.

Expressed quantitatively, the selectivity of the artificial serine proteases should be characterized by the value of (k(I)/k(V) being at most 0.06, and the value k(IIl)/k(V) being at most 0.5. It is preferred that (k(I)/k(V) is at most 0.05 and k(III)/k(V) is at most 0.4, and more preferred that (k(I)/k(V) is at most 0.04 and k(III)/k(V) is at most 0.15.

A more comprehensive specificity characterization involves further model substrates: thus, the substrate specificity could be assessed to be identical to or better than that of bovine blood coagulation factor X_a by each of the ratios (k(I)/k(V), k(II)/k(V), k(III)/k(V) and k(IV)/k(V)) between cleavage rate against each of the substrates I-IV:

I:

Benzoyl-Val-Gly-Arg-paranitroanilide, Tosyl-Gly-Pro-Lys-paranitroanilide, Tosyl-Gly-Pro-Arg-paranitroanilide, (d,1)Val-Leu-Arg-paranitroanilide

II:

IV:

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<img file="CA2155335C_D0009.tif" />

versus that against the substrate

V:

Benzoyl-lie-Glu-Gly-Arg-paranitroanilide at 20°C, pH=8 in a buffer consisting of 50 mM Tris, 100 mM NaCl, 1 mM CaCl₂, being identical to or lower than the corresponding ratio determined for bovine coagulation factor X_a which is substantially free from contaminating proteases .

Within this characterization, (k(I)/k(V) should be at most 0.06, k(II)/k(V) should be at most 0.03, k(III)/k(V) should be at most 0.5, and k(IV)/k(V)) should be at most 0.01, and it is preferred that (k(I)/k(V) is at most 0.05, k(II)/k(V) is at most 0.025, k(III)/k(V) is at most 0.4, and k(IV)/k(V)) is at most 0.008, and more preferred that (k(I)/k(V) is at most 0.04, k(II)/k(V) is at most 0.015, k(III)/k(V) is at most 0.15, and k(IV)/k(V)) is at most 0.005.

The serine protease type polypeptide as defined above will normally have a molecular weight, M^-, of at most 70,000 and at least 15,000.

One such novel polypeptide according to the invention has the amino acid sequence SEQ ID NO: 2 or is an analogue and/or homologue thereof. Other important embodiments of the polypeptide of the invention have an amino acid sequence which is a subsequence of SEQ ID NO: 2 or an analogue and/or homologue of such a subsequence.

By the use of the term an analogue of a polypeptide encoded by the DNA sequence or an analogue of a polypeptide having the amino acid sequence is meant any polypeptide which is capable of performing as bovine coagulation factor X_a in the tests mentioned above. Thus, included are also polypeptides from different sources, such as different mammals or vertebrates, which vary e.g. to a certain extent in the amino acid composition, or the post-translational modifications

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e.g. glycosylation or phosphorylation, as compared to the artificial serine protease described in the examples.

The term analogue is thus used in the present context to indicate a protein or polypeptide of a similar amino acid composition or sequence as the characteristic amino acid sequence SEQ ID NO: 2 derived from a artificial serine protease as described in Example 5, allowing for minor variations that alter the amino acid sequence e.g. deletions, site directed mutations, insertions of extra amino acids, or combinations thereof, to generate artificial serine protease analogues.

Therefore, in the present description and claims, an analogue (of a polypeptide) designates a variation of the polypeptide in which one or several amino acids may have been deleted or exchanged, and/or amino acids may have been introduced, provided the enzymatic activity with the above-defined specificity is retained, as can be assessed as described above.

With respect to homology, an analogue of a polypeptide according to the invention may have a sequence homology at the polypeptide level of at least 60% identity compared to the sequence of a fragment of SEQ ID NO: 2, allowing for deletions and/or insertions of at most 50 amino acid residues.

Such polypeptide sequences or analogues thereof which has a homology of at least 60% with the polypeptide shown in SEQ ID

NO: 2 encoded for by the DNA sequence of the invention SEQ ID NO: 1 or analogues and/or homologues thereof, constitute an important embodiment of this invention.

By the term sequence homology is meant the identity in sequence of either the amino acids in segments of two or more amino acids in a amino acid sequence, or the nucleotides in segments of two or more nucleotides in a nucleotide sequence. With respect to polypeptides, the terms are thus intended to mean a homology between the amino acids in question between

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PCT/DK94/00054 which the homology is to be established, in the match with respect to identity and position of the amino acids of the polypeptides .

The term homologous is thus used here to illustrate the 5 degree of identity between the amino acid sequence of a given polypeptide and the amino acid sequence shown in SEQ ID NO:

2. The amino acid sequence to be compared with the amino acid sequence shown in SEQ ID NO: 2 may be deduced from a nucleotide sequence such as a DNA or RNA sequence, e.g.

obtained by hybridization as defined in the following, or may be obtained by conventional amino acid sequencing methods.

Another embodiment relates to a polypeptide having an amino acid sequence from which a consecutive string of 20 amino acids is homologous to a degree of at least 40% with a string of amino acids of the same length selected from the amino acid sequence shown in SEQ ID NO: 2.

One serine protease polypeptide according to the invention has the amino acid sequence of SEQ ID NO: 2, residues 82-484, or is an analogue and/or homologue thereof. Another serine protease polypeptide according to the invention has the amino acid sequence of SEQ ID NO: 2, residues 166-484, or is an analogue and/or homologue thereof.

A number of modifications of the sequences shown herein are particularly interesting: The insertion of the cleaving directing sequences SEQ ID NO: 38 or 40-42 instead of residues 230-233 in SEQ ID NO: 2, combined with exchange of cysteine residue 245 by preferably Gly, Ser or Arg in SEQ ID NO: 2. Another interesting possibility is insertion of SEQ ID NO: 38 or 40-42 instead of residues 179-182 in SEQ ID NO: 2.

Quite generally, in any of the artificial serine proteases defined above, replacement of the cleaving sequence corresponding to residues 230-233 in SEQ ID NO: 2 v/ith one of the cleavage-directing sequences defined above will give rise to extremely useful cleaving enzymes for use in the method

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PCT/DK94/00054 according to the invention, in that these can be selectively and very efficiently cleaved by enzymes having the specific enzymatic activity of bovine coagulation factor X_a, and thus by artificial serine proteases as defined above, including by molecules identical to themselves. The latter fact means that artificial serine proteases modified by such insertion of the specific cleaving-directing sequences can be extremely effectively activated, as the first molecules cleaved and activated will be able to cleave other molecules, thus starting a chain reaction.

As mentioned above, it is a most important feature that the artificial serine proteases can be produced by recombinant DNA techniques, and hence, another important embodiment of the invention relates to a nucleic acid fragment capable of encoding a polypeptide according as defined above, in particular a DNA fragment which is capable of encoding an artificial serine protease polypeptide as defined above.

In one of its aspects, the invention relates to a nucleotide sequence encoding a polypeptide of the invention as defined above. In particular, the invention relates to a nucleotide sequence having the nucleotide sequence shown in the DNA sequence SEQ ID NO: 1 or an analogue thereof which has a homology with the any of the DNA sequences shown in SEQ ID NO: 1 of at least 60%, and/or encodes a polypeptide, the amino acid sequence of which is at least 60% homologous with the amino acid sequences shown in SEQ ID NO: 2.

Generally, only coding regions are used when comparing nucleotide sequences in order to determine their internal homology.

The term analogue with regard to the DNA fragments of the invention is intended to indicate a nucleotide sequence which encodes a polypeptide identical or substantially identical to the polypeptide encoded by a DNA fragment of the invention.

It is well known that the same amino acid may be encoded by

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PCT/DK94/00054 various codons-, the codon usage being related, inter alia, to the preference of the organisms in question expressing the nucleotide sequence. Thus, one or more nucleotides or codons of the DNA fragment of the invention may be exchanged by others which, when expressed, result in a polypeptide identical or substantially identical to the polypeptide encoded by the DNA fragment in question.

Furthermore, the term analogue is intended to allow for variations in the sequence such as substitution, insertion (including introns), addition and rearrangement of one or more nucleotides, which variations do not have any substantially effect on the polypeptide encoded by the DNA fragment.

Thus, within the scope of the present invention is a modified nucleotide sequence which differs from the DNA sequence shown in SEQ ID NO: 1 in that at least one nucleotide has been substituted, added, inserted, deleted and/or rearranged.

The term substitution is intended to mean the replacement of one or more nucleotides in the full nucleotide sequence with one or more different nucleotides, addition is under20 stood to mean the addition of one or more nucleotides at either end of the full nucleotide sequence, insertion is intended to mean the introduction of one or more nucleotides within the full nucleotide sequence, deletion is intended to indicate that one or more nucleotides have been deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it, and rearrangement is intended to mean that two or more nucleotide residues have been exchanged within the DNA or polypeptide sequence, respectively. The DNA fragment may, however, also be modified by mutagenesis either before or after inserting it in the organism. The DNA or protein sequence of the invention may be modified in such a way that it does not lose any of its biophysical, biochemical or biological properties, or part of such properties (one and/or all) or all of such properties (one and/or all).

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An example of a specific analogue of the DNA sequence of the invention is a DNA sequence which comprises the DNA sequence shown in SEQ ID NO: 1 and particularly adapted for expression in E. coli. This DNA sequence is one which, when inserted in

E. coli together with suitable regulatory sequences, results in the expression of a polypeptide having substantially the amino acid sequence shown in SEQ ID NO: 2. Thus, this DNA sequence comprises specific codons recognized by E. coli.

The terms fragment, sequence, homologue and analogue, 10 as used in the present specification and claims with respect to fragments, sequences, homologues and analogues according to the invention should of course be understood as not comprising these phenomena in their natural environment, but rather, e.g., in isolated, purified, in vitro or recombinant form.

One embodiment of the nucleic acid fragment according to the invention is a nucleic acid fragment as defined above in which at least 60% of the coding triplets encode the same amino acids as a nucleic acid fragment of the nucleic acid which encodes bovine coagulation factor X, allowing for insertions and/or deletions of at most 150 nucleotides. An example of such a nucleic acid fragment is SEQ ID NO: 1, nucleotides 76-1527, and analogues and/or homologues thereof. Another example is SEQ ID NO: 1, nucleotides 319-1527, and analogues and/or homologues thereof. Still another example is SEQ ID NO: 1, nucleotides 571-1527, and analogues and/or homologues thereof.

The DNA fragment described above and constituting an important aspect of the invention may be obtained directly from the genomic DNA or by isolating mRNA and converting it into the corresponding DNA sequence by using reverse transcriptase, thereby producing a cDNA. When obtaining the DNA fragment from genomic DNA, it is derived directly by screening for genomic sequences as is well known for the person skilled in the art. It can be accomplished by hybridization to a DNA

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PCT/DK94/00054 probe designed on the basis of knowledge of the sequences of the invention, or the sequence information obtained by amino acid sequencing of a purified serine protease. When the DNA is of complementary'' DNA (cDNA) origin, it may be obtained by preparing a cDNA library with mRNA from cells containing an artificial serine protease. Hybridization can be accomplished by a DNA probe designed on the basis of knowledge of the cDNA sequence, or the sequence information obtained by amino acid sequencing of a purified artificial serine protease.

The DNA fragment of the invention or an analogue and/or homologue thereof of the invention can be replicated by fusing it with a vector and inserting the complex into a suitable microorganism or a mammalian cell line. Alternatively, the DNA fragment can be manufactured using chemical synthesis. Also, polymerase chain reaction (PCR) primers can be synthesized based on the nucleotide sequence shown in SEQ ID NO: 1. These primers can then be used to amplify the whole or a part of a sequence encoding an artificial serine protease polypeptide.

Suitable polypeptides of the invention can be produced using recombinant DNA technology. More specifically, the polypeptides may be produced by a method which comprises culturing or breeding an organism carrying the DNA sequence shown in SEQ ID NO: 1 or an analogue and/or homologue thereof of the invention under conditions leading to expression of said DNA fragment, and subsequently recovering the expressed polypeptide from the said organism.

The organism which is used for the production of the polypeptide may be a higher organism, e.g. an animal, or a lower organism, e.g. a microorganism. Irrespective of the type of organism used, the DNA fragment of the invention (described above) should be introduced in the organism either directly or with the help of a suitable vector. Alternatively, the polypeptides may be produced in the mammalian cell lines by introducing the DNA fragment or an analogue and/or homologue

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PCT/DK94/00054 .2155335 thereof of the invention either directly or with the help of an expression vector.

The DNA fragment of the invention can also be cloned in a suitable stable expression vector and then put into a suit5 able cell line. The cells expressing the desired polypeptides are then selected using the conditions suitable for the vector and the cell line used. The selected cells are then grown further and form a very important and continuous source of the desired polypeptides.

Thus, another aspect of the invention relates to an expression system comprising a nucleic acid fragment as defined above and encoding an artificial serine protease polypeptide as defined above, the system comprising a 5'-flanking sequence capable of mediating expression of said nucleic acid fragment. The expression system may be a replicable expression vector carrying the nucleic acid fragment, which vector is capable of replicating in a host organism or a cell line; the vector may, e.g., be a plasmid, phage, cosmid, minichromosome or virus; the vector may be one which, when intro20 duced in a host cell, is integrated in the host cell genome.

Another aspect of the invention relates to an organism which carries and is capable of replicating the nucleic acid fragment as defined above. The organism may be a microorganism such as a bacterium, a yeast, a protozoan, or a cell derived from a multicellular organism such as a fungus, an insect cell, a plant cell, a mammalian cell or a cell line. Particularly interesting host organisms are microorganisms such as a bacterium of the genus Escherichia, Bacillus or Salmonella.

A further aspect of the invention relates to a method of producing an artificial serine protease polypeptide as defined above, comprising the following steps of:

1. inserting a nucleic acid fragment as defined above in an expression vector,

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2. transforming a host organism as defined above with the vector produced in step a,

3. culturing the host organism produced in step b to express the polypeptide,

4. harvesting the polypeptide,

5. optionally subjecting the polypeptide to posttranslational modification,

6. if necessary subjecting the polypeptide to the denaturing/renaturing cycling method according to the present invention, and

7. optionally subjecting the polypeptide to further modification to obtain an authentic polypeptide as defined above.

Further modifications of the polypeptides may for instance be 15 accomplished by subjecting the polypeptide molecules to carboxypeptidase A or B, whereby selected amino acid residues may be removed from the C-terminus of the polypeptide molecules. This is desirable under circumstances wherein the optimal folding of the authentic polypeptide molecules only is achieved when the N-terminus is free and the cleavage directing polypeptide (such as SEQ ID NO: 37) thus is placed C-terminally of the authentic polypeptide. As is known, carboxypeptidase B cleaves sequentially from the C-terminus, and only cleaves off basic amino acids, whereas carboxypepti25 dase A cleaves off non-basic amino acids. By careful designing which residue is adjoined C-terminally to the authentic polypeptide it is possible to ensure that all but the authentic polypeptide is cleaved by the carboxypeptidases. If the C-terminus of the authentic polypeptide is a basic amino acid residue one should assure that the C-terminally linked residue which is to be removed is non-basic and vice versa. If one knows the sequence of the amino acid residues from the CWO 94/18227 2155335 PCT/DK94/00054 terminus to the C-terminus of the authentic polypeptide it is possible to alternate between treatments with the two carboxypeptidases until only the naked, authentic polypeptide is left. A practical embodiment would be to use immobilized carboxypeptidases.

The polypeptide produced may be isolated by a method comprising one or more steps like affinity chromatography using immobilized polypeptide or antibodies reactive with said polypeptide and/or other chromatographic and electrophoretic procedures.

Also, it will be understood that a polypeptide of the invention may be prepared by the well known methods of liquid or solid phase peptide synthesis utilizing the successive coupling of the individual amino acids of the polypeptide sequence. Alternatively, the polypeptide can be synthesized by the coupling of individual amino acids forming fragments of the polypeptide sequence which are later coupled so as to result in the desired polypeptide. These methods thus constitute another interesting aspect of the invention.

The invention also relates to the use of an artificial serine protease polypeptide as defined above for cleaving polypeptides at the cleavage site for bovine coagulation factor X_a, the cleavage site having the amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID

NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, and to the use of a an artificial serine protease polypeptide as defined above for cleaving polypeptides at the cleavage site for bovine coagulation factor X_a, the cleavage site having a modified version of the amino acid sequence selected from the group of

SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO:

46, which has been converted to a cleavable form as described further above.

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LEGENDS TO FIGURES

Fig. 1: Schematic representation of segment of a cyclic dénaturation / renaturation time-programme.

Solvent composition is expressed in terms of a binary mixture of a non-denaturing 'buffer A' and a denaturing 'buffer B' in terms of relative content of buffer B. Three consecutive cycles are represented, each consisting of a renaturation phase 'F' and a dénaturation phase 'D'. Changes in level of denaturing power of the solvent mixture during dénaturation phases in consecutive cycles are denoted 'k'.

Fig. 2: Construction of the expression plasmids pT₇H₆FX-h/?2m and pT₇H₆FX-m£2m.

The amplified DNA fragments containing the reading frames of human- and murine β₂-microglobulin from amino acid residues Ile-L to Met₉₉, fused at the 5'-end to the nucleotide sequences encoding the FX_a cleavage site (SEQ ID NO: 37), were cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pT₇H₆ using standard procedures.

Fig. 3: Amino acid sequences of human- and murine |S₂-microglobulin.

A: Predicted amino acid sequence of the full length reading frame encoding human β₂-microglobulin (SEQ ID NO: 49) . Amino acid residue one (lie) in the processed mature protein is indicated. B: Predicted amino acid sequence of the full length reading frame encoding murine 0₂-microglobulin (SEQ ID NO: 50). Amino acid residue one (lie) in the processed mature protein is indicated.

Fig. 4: Construction of the expression plasmid pT₇H₆FX-hGH. The amplified DNA fragment containing the reading frame of human Growth Hormone from amino acid residues Phe_x to Phe₁₉₁, fused at the 5'-end to the nucleotide sequence encoding the FX_a cleavage site IEGR (SEQ ID NO: 38), was cut with the

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PCT/DK94/00054 restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄ DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pT₇H₆ using standard procedures.

Fig. 5: Amino acid sequence of human Growth Hormone (Somatotropin) .

The predicted amino acid sequence of the full length reading frame encoding human Growth Hormone (SEQ ID NO: 51). The first Amino acid residue in the processed mature protein (Phe-J is indicated.

Fig. 6: Construction of the plasmids pT₇H₆FX-#l, #2, and #3 expressing amino acid residue no. 20 (Ala) to 109 (Arg), amino acid residue no 20 (Ala) to 190 (Ala), and amino acid residue no. 20 (Ala) to 521 (Lys) of the human a₂-Macroglobu15 lin Receptor Protein (a₂MR) (SEQ ID NO: 52).

The amplified DNA fragments derived from the reading frame of the a₂MR from #1: amino acid residue no. 20 (Ala) to 109 (Arg), #2: amino acid residue no. 20 (Ala) to 190 (Ala), and #3: amino acid residue no. 20 (Ala) to 521 (Lys) , fused at the 5'-end to the nucleotide sequence encoding the FX_a cleavage site IEGR (SEQ ID NO: 38), were cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄ DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pT₇H₆ using standard procedures.

Fig. 7: Construction of the plasmids pLcIIMLCH₆FX-#4, #5, and #6 expressing amino acid residue no. 803 (Gly) to 1265 (Asp), amino acid residue no. 849 (Val) to 1184 (Gin), and amino acid residue no. 1184 (Gin) to 1582 (Lys) of the human a₂30 Macroglobulin Receptor Protein (a₂MR) (SEQ ID NO: 52) .

The amplified DNA fragments derived from the reading frame of the a₂MR from #4: amino acid residue no. 803 (Gly) to 1265 (Asp), #5: amino acid residue no. 849 (Val) to 1184 (Gin), and #6: amino acid residue no. 1184 (Gin) to 1582 (Lys), fused at the 5'-end to the nucleotide sequence encoding the

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215533b ₄₈

FX_a cleavage site IEGR (SEQ ID NO: 38), were cut with the restriction endonucleases Bam HI or Bel and Hind III (purchased from Boehringer, Germany) and ligated with T₄ DNA ligase (purchased from Boehringer, Germany) into Bam HI and

Hind III cut pLcIIMLCHgFX using standard procedures.

Fig. 8: Construction of the plasmids pLcIIMLCHgFX-#7, #8, and #9 expressing amino acid residue no. 803 (Gly) to 1582 (Lys), amino acid residue no. 2519 (Ala) to 2941 (lie), and amino acid residue no. 3331 (Val) to 3778 (lie) of the human a₂10 Macroglobulin Receptor Protein (œ₂MR) (SEQ ID NO: 52) .

The amplified DNA fragments derived from the reading frame of the q;₂MR from #7: amino acid residue no. 803 (Gly) to 1582 (Lys), #8: amino acid residue no. 2519 (Ala) to 2941 (lie), and #9: amino acid residue no. 3331 (Val) to 3778 (lie), fused at the 5'-end to the nucleotide sequence encoding the FX_a cleavage site IEGR (SEQ ID NO: 38), were cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄ DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pLcIIMLCHgFX using standard procedures.

Figs. 9a and 9b: Amino acid sequence of human a₂-Macroglobulin Receptor Protein (a₂MR) (SEQ ID NO: 52).

The predicted amino acid sequence of the full length reading frame encoding the a₂MR. Amino acid residues present in the recombinant proteins as N- or C-terminal residues are identified by their numbers above the œ₂MR sequence.

Fig. 10: Construction of the expression plasmid pLcIIMLCHgFXFXAy.

The amplified DNA fragment containing the reading frame of 30 bovine blood coagulation Factor X from amino acid residue

Ser₈₂ to Trp_4a4, (FXAy) fused at the 5'-end to the nucleotide sequence encoding the FX_a cleavage site IEGR (SEQ ID NO: 38), was cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄

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4S

DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pLcIIMLCHgFX using standard procedures.

Fig. 11: Amino acid sequence of bovine blood coagulation Factor X (FX).

The predicted amino acid sequence of the full length reading frame encoding bovine FX (SEQ ID NO: 53). The N-terminal amino acid residue Ser₈₂ and the C-terminal Trp₄₈₄ residue in the FXAy construct are identified.

Fig. 12: Construction of the expression plasmid pLcIIMLCHgFX10 Kl.

The amplified DNA fragment containing the reading frame of human plasminogen kringle 1 (Kl) from amino acid residue Ser₈₂ to G1u₁₆₂ (numbering as in Glu-plasminogen) , fused at the 5'-end to the nucleotide sequence encoding the FX_a clea15 vage site IEGR (SEQ ID NO: 38), was cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄ DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pLcIIMLCHgFX using standard procedures.

Fig. 13: Construction of the expression plasmid pLcIIH₆FX~K4.

The amplified DNA fragment containing the reading frame of human plasminogen kringle 4 (K4) from amino acid residue Val₃₅₄ to Ala₄₃₉ (numbering as in Glu-plasminogen) , fused at the 5'-end to the nucleotide sequence encoding the FX_a clea25 vage site IEGR (SEQ ID NO: 38), was cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄ DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pLcIIH₆FX using standard procedures.

Fig. 14: Amino acid sequence of human Glu- Plasminogen (SEQ ID NO: 54). The N- and C-terminal amino acid residues in the Kl and K4 constructs are identified by their numbers in the sequence.

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Fig. 15: SDS-PAGE analysis of production and in vitro folding of recombinant human β₂-microglobulin.

Lane 1: Crude protein extract before application to the Ni²⁺NTA-agarose column (reduced sample).

Lane 2: Column flow-through during application of the crude protein extract onto the Ni²⁺NTA-agarose column (reduced sample)

Lane 3: Human £₂-microglobulin eluted from the Ni²⁺NTA-agarose column after the cyclic folding procedure by the non10 denaturing elution buffer (reduced sample).

Lane 4: Protein markers (Pharmacia, Sweden): From top of gel; 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa, and 14.4 kDa (reduced sample)

Lane 5: Same as lane 3 (non-reduced sample)

Lane 6: Recombinant human 0₂-microglobulin after FX_a cleavage and final purification (non-reduced sample).

Fig. 16: SDS-PAGE analysis of in vitro folding of recombinant human Growth Hormone; hGH (Somatotropin).

Lane 1: Protein markers (Pharmacia, Sweden): From top of gel; __ 20 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa, and 14.4 kDa (reduced sample)

Lane 2: Human hGH eluted from the Ni²⁺NTA-agarose column after the cyclic folding procedure by the non-denaturing elution buffer (non-reduced sample).

Lane 3: Human hGH eluted from the Ni²⁺NTA-agarose column after the cyclic folding procedure by the denaturing elution buffer B from the folding procedure (non-reduced sample).

Lane 4-18: Fractions collected during the separation of monomeric hGH-fusion protein from dimer and multimer fusion proteins after the cyclic folding procedure by ion exchange

-ΠΛ chromatography on Q-Sepharose (Pharmacia, Sweden). The monomeric protein was eluted in a peak well separated from the peak containing the dimer and multimer proteins (nonreduced samples).

Fig. 17: SDS-PAGE analysis of in vitro folding of recombinant kringle 1 and 4 from human plasminogen and recombinant fusion

WO 94/18227 2155335 PCT/DK94/00054 protein #4 derived from human œ₂-Macroglobulin Receptor Protein (c^MR) ·

Lane 1: Protein markers {Pharmacia, Sweden): From top of gel; 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa, and 14.4 kDa (reduced sample).

Lane 2: Crude Kl-fusion protein extract before application to the Ni²⁺NTA-agarose column (reduced sample).

Lane 3: Kl-fusion protein eluted from the Ni²⁺NTA-agarose column after the cyclic folding procedure by the non-denatu10 ring elution buffer (reduced sample).

Lane 4: Same as lane 3 (non-reduced sample).

Lane 5: Flow-through from the lysine-agarose column during application of the Kl-fusion protein (non-reduced sample). Lane 6: Kl-fusion protein eluted from the lysine-agarose column (non-reduced sample).

Lane 7: K4-fusion protein eluted from the Ni²⁺NTA-agarose column after the cyclic folding procedure by the non-denaturing elution buffer (reduced sample).

Lane 8: Same as lane 7 (non-reduced sample).

Lane 9: œ₂MR#4 fusion protein eluted from the Ni²+NTA-agarose column after the cyclic folding procedure by the non-denaturing elution buffer (reduced sample).

Lane 10: Same as lane 9 (non-reduced sample).

Fig. 18: Construction of the expression plasmid pT₇H₆FX2 5 a₂MRBDv.

The amplified DNA fragment containing the reading frame of human a₂-Macroglobulin from amino acid residues Val₁₂₉₉ to Ala₁₄₅₁, fused at the 5'-end to the nucleotide sequence encoding the FX_a cleavage site IEGR (SEQ ID NO: 38), was cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄ DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pT₇H₆ using standard procedures.

Fig. 19: Amino acid sequence of the receptor-binding domain of human a₂-Macroglobulin (from residue Val₁₂₉₉ to Ala₁₄₅₁) (SEQ ID NO: 55).

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Fig. 20: Construction of the expression plasmid pT₇H₆FX-TETN. The amplified DNA fragment containing the reading frame of mature monomeric human Tetranectin from amino acid residues GIu-l to Val₁₈₁, fused at the 5'-end to the nucleotide sequence encoding the FX_a cleavage site IEGR (SEQ ID NO: 38), was cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pT₇H₆ using standard procedures.

Fig. 21: Amino acid sequence of human monomeric Tetranectin. The predicted amino acid sequence of the full length reading frame encoding human Tetranectin (SEQ ID NO: 56). The first Amino acid residue in the processed mature protein (Glu-J is indicated.

Fig. 22: Construction of the expression plasmid pT₇H₆FX-DB32. The amplified DNA fragment containing the reading frame of the artificial diabody DB32 from amino acid residues Gln_x to Asn₂₄₆, fused at the 5'-end to the nucleotide sequence encoding the FX_a cleavage site IEGR (SEQ ID NO: 38), was cut with the restriction endonucleases Bam HI and Hind III (purchased from Boehringer, Germany) and ligated with T₄ DNA ligase (purchased from Boehringer, Germany) into Bam HI and Hind III cut pT₇H₆ using standard procedures.

Fig. 23: Amino acid sequence of the artificial diabody DB32 (SEQ ID NO: 57).

Fig. 24: The expression plasmid pT₇H₆FX-PS.4.

The construction of pT₇H₆FX-PS.4 expressing human psoriasin from amino acid residues Ser₂ to Gln₁₀₁ has previously been described (Hoffmann, 1994).

Fig. 25: Amino acid sequence of human psoriasin.

The predicted amino acid sequence of the full length reading frame encoding human psoriasin (SEQ ID NO: 58).

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Fig. 26: SDS-PAGE analysis of purification and FX_a cleavage of recombinant Mab 32 diabody. a: Different stages of the purification Lanes 1 and 2: Crude product from folding.

Lane 3: Final purified Mab 32 diabody fusion protein product Lane 4: Supernatant of crude folding product after 50-fold concentration and centrifugation.

Lane 5: Pellet from crude folding product after 50-fold concentration and centrifugation.

b: FX_a cleavage of Mab 32 diabody fusion protein.

Lanes 1 and 5: Final purified Mab 32 diabody fusion protein

<td> Lane</td><td> 2: Molar ratio</td><td> 1:5</td><td> FX_a:Mab</td><td> 32</td><td> diabody</td><td> fusion</td><td> protein</td><td> at</td>

<td> 37°C</td><td> for 20 hours</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Lane</td><td> 3: Molar ratio</td><td> 1:2</td><td> FX_a:Mab</td><td> 32</td><td> diabody</td><td> fusion</td><td> protein</td><td> at</td>

<td> 37°C</td><td> for 20 hours</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Lane</td><td> 4: Molar ratio</td><td> 1:1</td><td> FX_a:Mab</td><td> 32</td><td> diabody</td><td> fusion</td><td> protein</td><td> at</td>

37°C for 20 hours

Fig 27: Suitability of glutathione as reducing agent in cyclic refolding of human β₂-microglobulin fusion protein.

<td> 20</td><td> Lane</td><td> 1:</td><td colspan="3"> Reduced sample of test</td><td> no.</td><td> 1.</td>

<td></td><td> Lane</td><td> 2:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.l.</td>

<td></td><td> Lane</td><td> 3:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.2 .</td>

<td></td><td> Lane</td><td> 4:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 3 .</td>

<td></td><td> Lane</td><td> 5 :</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 4.</td>

<td> 25</td><td> Lane</td><td> 6:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 5.</td>

<td></td><td> Lane</td><td> 7:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 6.</td>

<td></td><td> Lane</td><td> 8:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 7.</td>

<td></td><td> Lane</td><td> 9:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 8 .</td>

Lane 10: Non-reduced sample of test no.9.

Lane 11: Non-reduced sample of test no.10.

Lane 12: Non-reduced sample of test no.11.

Fig. 28: Suitability of L-cysteine ethyl ester as reducing agent in cyclic refolding of human £₂^m^^cr°9^ioimlin fusion protein.

Lane 1: Reduced sample of test no. 1.

Lane 2: Non-reduced sample of test no.l.

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<td> Lane</td><td> 3:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.2 .</td>

<td> Lane</td><td> 4 :</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 3.</td>

<td> Lane</td><td> 5:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 4 .</td>

<td> Lane</td><td> 6:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 5 .</td>

<td> Lane</td><td> 7:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 6 .</td>

<td> Lane</td><td> 8:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 7.</td>

<td> Lane</td><td> 9 :</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 8 .</td>

Lane 10: Non-reduced sample of test no.9

<td colspan="3"> Reduced sample of test</td><td> , no.</td><td> 1.</td>

<td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 1.</td>

<td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.2 .</td>

<td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 3 .</td>

<td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.4 .</td>

<td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.5 .</td>

<td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 6.</td>

<td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 7.</td>

<td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 8.</td>

Fig. 29: Suitability of 2-Mercaptoethanol as reducing agent 10 in cyclic refolding of human 0₂‘^mi^cro9l^ok^u^ⁿ fusion protein.

Lane l Lane 2 Lane 3 Lane 4 15 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9

Lane 10: Non-reduced sample of test no.9.

Fig. 30: Suitability of Mercaptosuccinic acid as reducing agent in cyclic refolding of human β₂ -microglobulin fusion protein.

<td></td><td> Lane</td><td> 1:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.l.</td>

<td> 25</td><td> Lane</td><td> 2:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.2 .</td>

<td></td><td> Lane</td><td> 3 :</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 3.</td>

<td></td><td> Lane</td><td> 4:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.4 .</td>

<td></td><td> Lane</td><td> 5:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 5 .</td>

<td></td><td> Lane</td><td> 6:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.6 .</td>

<td> 30</td><td> Lane</td><td> 7:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 7.</td>

<td></td><td> Lane</td><td> 8 :</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 8 .</td>

<td></td><td> Lane</td><td> 9:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 9 .</td>

Fig. 31: Suitability of N-Acetyl-L-cysteine as reducing agent in cyclic refolding of human fusion protein.

Lane 1: Reduced sample of test no. 1.

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<td> Lane</td><td> 2:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. l.</td>

<td> Lane</td><td> 3 :</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.2 .</td>

<td> Lane</td><td> 4:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 3 .</td>

<td> Lane</td><td> 5:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no.4 .</td>

<td> Lane</td><td> 6:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 5 .</td>

<td> Lane</td><td> 7:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 6 .</td>

<td> Lane</td><td> 8:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 7 .</td>

<td> Lane</td><td> 9:</td><td> Non-reduced</td><td> sample</td><td> of</td><td> test</td><td> no. 8 .</td>

Lane 10: Non-reduced sample of test no.9.

Fig. 32: SDS-PAGE analysis of cyclic refolding of human β₂microglobulin fusion protein.

Lane 1: Crude protein extract before application to the Ni²⁺NTA-agarose column (reduced sample).

<td> Lane 2: 8</td><td> μΐ</td><td> sample</td><td> of</td><td> soluble</td><td> fraction</td><td> of</td><td> refolded</td><td> h/3₂m</td><td> as</td>

<td> described</td><td> in</td><td> EXAMPLE</td><td> 1.</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Lane 3: 4</td><td> μΐ</td><td> sample</td><td> of</td><td> soluble</td><td> fraction</td><td> of</td><td> refolded</td><td> h/3₂m</td><td> as</td>

<td> described</td><td> in</td><td> EXAMPLE</td><td> ; 1.</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Lane 4: 2</td><td> μΐ</td><td> sample</td><td> of</td><td> soluble</td><td> fraction</td><td> of</td><td> refolded</td><td> h0₂m</td><td> as</td>

described in EXAMPLE 1.

0 Lane 5: 8 μΐ sample of insoluble fraction of refolded h0₂m as described in EXAMPLE 1.

Lanes 6 and 7: hj3₂^m final product after purification by ion exchange chromatography.

Lanes 8 and 9: Refolded h0₂m after optimized refolding proto25 col as described in EXAMPLE 13.

Fig. 33: SDS-PAGE analysis of refolding of human β₂-microglobulin fusion protein by buffer step and linear gradient. Lane 1: Sample from soluble fraction of refolded h0₂m, folded by the buffer step protocol as described in EXAMPLE 13.

Lane 2 and 3: Sample of insoluble fraction of refolded h/3₂m, folded by the buffer step protocol as described in EXAMPLE 13.

Lane 4: Protein molecular weight markers (Pharmacia, Sweden) .From top of gel; 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa, and 14.4 kDa (reduced sample).

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Lane 5: Sample of soluble fraction of refolded h0₂m, folded by the linear gradient protocol as described in EXAMPLE 13 Lane 6 and 7: Sample of insoluble fraction of refolded h/3₂m, folded by the linear gradient protocol as described in

EXAMPLE 13.

Fig. 34: The general scheme of the design of the fusion proteins described in the examples.

In the N-terminal end of the fusion protein is optionally inserted a booster segment enhancing the level of exprès10 sion of the fusion protein in the cell expressing the DNA encoding the fusion protein. C-terminally to this, the 6H indicates the 6 histidinyl residues which constitute an ion chelating site used as a affinity handle during purification and refolding of the fusion proteins. The FX at the C terminal of the 6 histidinyl site is the FX_a cleavage site. Finally, the part of the fusion protein denoted protein represents the protein which is going to be refolded according to the method of the invention.

EXAMPLES

Examples 1 to 11 given in this section, which are used to exemplify the cyclic folding procedure, all describe the process of folding a recombinant cleavable hybrid protein (fusion protein) produced in E. coli, purified from a crude protein extract and subjected to folding without further purification by one general procedure.

The nucleotide sequence encoding the recombinant protein, which is to be produced, is at the 5'-end fused to a nucleotide sequence encoding an amino acid sequence specifying a FX_a cleavage site (FX), in turn linked N-terminally to a segment containing six histidinyl residues (SEQ ID NO: 47). The linking of the FX_a cleavage site is normally achieved during a Polymerase Chain Reaction, wherein the 5'-terminal primer comprises nucleotides encoding this sequence. The linking of the six histidinyl residues is normally obtained

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PCT/DK94/00054 by employing a vector which comprises a nucleotide fragment encoding SEQ ID NO: 47. The six histidinyl residues constitute a metal ion chelating site, which is utilized as affinity handle during purification of the fusion protein and subsequently as the point of contact to the solid matrix during the cyclic folding process. Occasionally 'booster segments' (e.g. a segment derived from the N-terminus of the XcII protein in some cases followed by a segment derived from myosin light chain) are inserted N-terminal to the affinity handle in order to improve the level of expression of the fusion protein in £. coli.

The fusion proteins are all designed according to the same general scheme (cf. fig. 34). The presence of booster segments, affinity handle and FX_a cleavage site might complicate refolding of the recombinant protein of interest. Furthermore, the cyclic folding process is initiated immediately after the affinity purification of the fusion protein. This means that fusion protein material, which have been partially degraded by the E. coll host is retained on the affinity matrix in addition to the full length fusion protein column. This degraded fusion protein may well interfere severely with refolding of the full-length fusion protein, thereby reducing the apparent efficiency of the process. The folding efficiency results reported in Examples 1 to 11 therefore cannot directly be compared to the efficiency of the process of refolding a purified fusion protein.

Examples 1 to 11 describe the refolding procedure for 21 different proteins, protein domains or domain-clusters, ranging from a size of 82 amino acids (Kl, Example 6) to 780 amino acids (œ₂MR#7, Example 4), and the number of disulphide bridges in the proteins ranges from zero (œ₂MRAP, Example 3) to 33 (a₂MR#4, Example 4) and 36 (a₂MR#7, Example 4) ,

The efficiency of the refolding of the proteins ranges from 15 to 95%, and the yield of active protein lies in the order

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PCT/DK94/00054 of 10-100 mg for refolding on a 40 ml Ni+NTA-agarose column (NTA denotes a substituted nitrilotriacetic acid).

The following tables 1-5 demonstrate the gradient profiles used in the examples. Time” is given in minutes and flow in ml/min.

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TABLE 1

<td> Step</td><td> Tme</td><td> Flow</td><td> %A</td><td> %B</td><td> Step</td><td> Tme</td><td> Flow</td><td> %A</td><td> HB</td>

<td> 1</td><td> 0</td><td> 2</td><td> 100</td><td> 0</td><td> 61</td><td> 900</td><td> 2</td><td> 100</td><td> 0</td>

<td> 2</td><td> 45</td><td> 2</td><td> 100</td><td> 0</td><td> 62</td><td> 945</td><td> 2</td><td> 100</td><td> 0</td>

<td> 3</td><td> 46</td><td> 2</td><td> 0</td><td> 100</td><td> 63</td><td> 946</td><td> 2</td><td> 60</td><td> 40</td>

<td> 4</td><td> 52</td><td> 2</td><td> 0</td><td> 100</td><td> 64</td><td> 952</td><td> 2</td><td> 60</td><td> 40</td>

<td> 5</td><td> 60</td><td> 2</td><td> 100</td><td> 0</td><td> 65</td><td> 960</td><td> 2</td><td> 100</td><td> 0</td>

<td> 6</td><td> 105</td><td> 2</td><td> 100</td><td> 0</td><td colspan="2"> 66 1005</td><td> 2</td><td> 100</td><td> 0</td>

<td> 7</td><td> 106</td><td> 2</td><td> 4</td><td> 96</td><td> 67</td><td> 1006</td><td> 2</td><td> 62</td><td> 38</td>

<td> 8</td><td> 113</td><td> 2</td><td> 4</td><td> 96</td><td> 68</td><td> 1012</td><td> 2</td><td> 62</td><td> 38</td>

<td> 9</td><td> 120</td><td> 2</td><td> 100</td><td> 0</td><td> 69</td><td> 1020</td><td> 2</td><td> 100</td><td> 0</td>

<td> 10</td><td> 165</td><td> 2</td><td> 100</td><td> 0</td><td> 70</td><td> 1065</td><td> 2</td><td> 100</td><td> 0</td>

<td> 11</td><td> 166</td><td> 2</td><td> 8</td><td> 92</td><td> 71</td><td> 1066</td><td> 2</td><td> 64</td><td> 36</td>

<td> 12</td><td> 172</td><td> 2</td><td> 8</td><td> 92</td><td> 72</td><td> 1072</td><td> 2</td><td> 64</td><td> 36</td>

<td> 13</td><td> 180</td><td> 2</td><td> 100</td><td> 0</td><td> 73</td><td> 1080</td><td> 2</td><td> 100</td><td> 0</td>

<td> 14</td><td> 225</td><td> 2</td><td> 100</td><td> 0</td><td> 74</td><td> 11Z5</td><td> 2</td><td> 100</td><td> 0</td>

<td> 15</td><td> 226</td><td> 2</td><td> 12</td><td> 88</td><td> 75</td><td> 1126</td><td> 2</td><td> 66</td><td> 34</td>

<td> 16</td><td> 232</td><td> 2</td><td> 12</td><td> 88</td><td> 76</td><td> 1132</td><td> 2</td><td> 66</td><td> 34</td>

<td> 17</td><td> 240</td><td> 2</td><td> 100</td><td> 0</td><td> 77</td><td> 1140</td><td> 2</td><td> 100</td><td> 0</td>

<td> 18</td><td> 285</td><td> 2</td><td> 100</td><td> 0</td><td> 78</td><td> 1185</td><td> 2</td><td> 100</td><td> 0</td>

<td> 19</td><td> 286</td><td> 2</td><td> 16</td><td> 84</td><td> 79</td><td> 1186</td><td> 2</td><td> 68</td><td> 32</td>

<td> 20</td><td> 292</td><td> 2</td><td> 16</td><td> 84</td><td> 80</td><td> 1192</td><td> 2</td><td> 68</td><td> 32</td>

<td> 21</td><td> 300</td><td> 2</td><td> 100</td><td> 0</td><td> 81</td><td> 1200</td><td> 2</td><td> 100</td><td> 0</td>

<td> 22</td><td> 345</td><td> 2</td><td> 100</td><td> 0</td><td> 82</td><td> 1245</td><td> 2</td><td> 100</td><td> 0</td>

<td> 23</td><td> 346</td><td> 2</td><td> 20</td><td> 80</td><td> 83</td><td> 1246</td><td> 2</td><td> 70</td><td> 30</td>

<td> 24</td><td> 352</td><td> 2</td><td> 20</td><td> 80</td><td> 64</td><td> 1252</td><td> 2</td><td> 70</td><td> 30</td>

<td> 25</td><td> 360</td><td> 2</td><td> 100</td><td> 0</td><td> 85</td><td> 1260</td><td> 2</td><td> 100</td><td> 0</td>

<td> 26</td><td> 405</td><td> 2</td><td> 100</td><td> 0</td><td colspan="2"> 86 1305</td><td> 2</td><td> 100</td><td> 0</td>

<td> 27</td><td> 406</td><td> 2</td><td> 24</td><td> 76</td><td> 87</td><td> 1306</td><td> 2</td><td> 72</td><td> 28</td>

<td> 28</td><td> 412</td><td> 2</td><td> 24</td><td> 76</td><td> 88</td><td> 1312</td><td> 2</td><td> 72</td><td> 28</td>

<td> 29</td><td> 420</td><td> 2</td><td> 100</td><td> 0</td><td> 89</td><td> 1319</td><td> 2</td><td> 100</td><td> 0</td>

<td> 30</td><td> 465</td><td> 2</td><td> 100</td><td> 0</td><td> 90</td><td> 1364</td><td> 2</td><td> 100</td><td> 0</td>

<td> 31</td><td> 466</td><td> 2</td><td> 28</td><td> 72</td><td> 91</td><td> 1365</td><td> 2</td><td> 74</td><td> 26</td>

<td> 32</td><td> 472</td><td> 2</td><td> 26</td><td> 72</td><td> 92</td><td> 1371</td><td> 2</td><td> 74</td><td> 26</td>

<td> 33</td><td> 480</td><td> 2</td><td> 100</td><td> 0</td><td> 93</td><td> 1378</td><td> 2</td><td> 100</td><td> 0</td>

<td> 34</td><td> 525</td><td> 2</td><td> 100</td><td> 0</td><td> 94</td><td> 1423</td><td> 2</td><td> 100</td><td> 0</td>

<td> 35</td><td> 526</td><td> 2</td><td> 32</td><td> 68</td><td> 95</td><td> 1424</td><td> 2</td><td> 76</td><td> 24</td>

<td> 36</td><td> 532</td><td> 2</td><td> 32</td><td> 68</td><td> 96</td><td> 1430</td><td> 2</td><td> 76</td><td> 24</td>

<td> 37</td><td> 540</td><td> 2</td><td> 100</td><td> 0</td><td> 97</td><td> 1437</td><td> 2</td><td> 100</td><td> 0</td>

<td> 38</td><td> 585</td><td> 2</td><td> 100</td><td> 0</td><td> 98</td><td> 1482</td><td> 2</td><td> 100</td><td> 0</td>

<td> 39</td><td> 586</td><td> 2</td><td> 36</td><td> 64</td><td> 99</td><td> 1483</td><td> 2</td><td> 78</td><td> 22</td>

<td> 40</td><td> 592</td><td> 2</td><td> 36</td><td> 64</td><td> 100</td><td> 1489</td><td> 2</td><td> 78</td><td> 22</td>

<td> 41</td><td> 600</td><td> 2</td><td> 100</td><td> 0</td><td> 101</td><td> 1496</td><td> 2</td><td> 100</td><td> 0</td>

<td> 42</td><td> 645</td><td> 2</td><td> 100</td><td> 0</td><td> 102</td><td> 1541</td><td> 2</td><td> 100</td><td> 0</td>

<td> 43</td><td> 646</td><td> 2</td><td> 40</td><td> 60</td><td> 103</td><td> 1542</td><td> 2</td><td> 80</td><td> 20</td>

<td> 44</td><td> 652</td><td> 2</td><td> 40</td><td> 60</td><td> 104</td><td> 1548</td><td> 2</td><td> 80</td><td> 20</td>

<td> 45</td><td> 660</td><td> 2</td><td> 100</td><td> 0</td><td> 105</td><td> 1555</td><td> 2</td><td> 100</td><td> 0</td>

<td> 46</td><td> 705</td><td> 2</td><td> 100</td><td> 0</td><td> 106</td><td> 1556</td><td> 2</td><td> 82</td><td> 18</td>

<td> 47</td><td> 706</td><td> 2</td><td> 44</td><td> 56</td><td> 107</td><td> 1562</td><td> 2</td><td> 82</td><td> 18</td>

<td> 48</td><td> 713</td><td> 2</td><td> 44</td><td> 56</td><td> 108</td><td> 1569</td><td> 2</td><td> 100</td><td> 0</td>

<td> 49</td><td> 720</td><td> 2</td><td> 100</td><td> 0</td><td> 109</td><td> 1614</td><td> 2</td><td> 100</td><td> 0</td>

<td> 50</td><td> 765</td><td> 2</td><td> 100</td><td> 0</td><td> 110</td><td> 1615</td><td> 2</td><td> 84</td><td> 16</td>

<td> 51</td><td> 766</td><td> 2</td><td> 48</td><td> 52</td><td> 111</td><td> 1621</td><td> 2</td><td> 84</td><td> 16</td>

<td> 52</td><td> 772</td><td> 2</td><td> 48</td><td> 52</td><td> 112</td><td> 1628</td><td> 2</td><td> 100</td><td> 0</td>

<td> 53</td><td> 780</td><td> 2</td><td> 100</td><td> 0</td><td> 113</td><td> 1673</td><td> 2</td><td> 100</td><td> 0</td>

<td> 54</td><td> 825</td><td> 2</td><td> 100</td><td> 0</td><td> 114</td><td> 1674</td><td> 2</td><td> 88</td><td> 12</td>

<td> 55</td><td> 826</td><td> 2</td><td> 52</td><td> 48</td><td> 115</td><td> 1732</td><td> 2</td><td> 88</td><td> 12</td>

<td> 56</td><td> 832</td><td> 2</td><td> 52</td><td> 48</td><td> 116</td><td> 1733</td><td> 2</td><td> 100</td><td> 0</td>

<td> 57</td><td> 840</td><td> 2</td><td> 100</td><td> 0</td><td> 117</td><td> 1778</td><td> 2</td><td> 100</td><td> 0</td>

<td> 58</td><td> 885</td><td> 2</td><td> 100</td><td> 0</td><td></td><td></td><td></td><td></td><td></td>

<td> 59</td><td> 886</td><td> 2</td><td> 56</td><td> 44</td><td></td><td></td><td></td><td></td><td></td>

<td> 60</td><td> 892</td><td> 2</td><td> 56</td><td> 44</td><td></td><td></td><td></td><td></td><td></td>

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TABLE 2

<td> Step</td><td> Time</td><td> Flow</td><td> %A</td><td> 968</td><td> Step</td><td> Time</td><td> Flow</td><td> %A</td><td> 968</td>

<td> 1</td><td> 0</td><td> 2</td><td> 100</td><td> 0</td><td> 49</td><td> 720</td><td> 2</td><td> 100</td><td> 0</td>

<td> 2</td><td> 45</td><td> 2</td><td> 100</td><td> 0</td><td> 50</td><td> 765</td><td> 2</td><td> 100</td><td> 0</td>

<td> 3</td><td> 46</td><td> 2</td><td> 0</td><td> 100</td><td> 51</td><td> 766</td><td> 2</td><td> 74</td><td> 26</td>

<td> 4</td><td> 52</td><td> 2</td><td> 0</td><td> 100</td><td> 52</td><td> . 772</td><td> 2</td><td> 74</td><td> 26</td>

<td> 5</td><td> 60</td><td> 2</td><td> 100</td><td> 0</td><td> 53</td><td> 780</td><td> 2</td><td> 100</td><td> 0</td>

<td> 6</td><td> 105</td><td> 2</td><td> 100</td><td> 0</td><td> 54</td><td> 825</td><td> 2</td><td> 100</td><td> 0</td>

<td> 7</td><td> 106</td><td> 2</td><td> 8</td><td> 92</td><td> 55</td><td> 826</td><td> 2</td><td> 76</td><td> 24</td>

<td> 8</td><td> 113</td><td> 2</td><td> 8</td><td> 92</td><td> 56</td><td> 832</td><td> 2</td><td> 76</td><td> 24</td>

<td> 9</td><td> 120</td><td> 2</td><td> 100</td><td> 0</td><td> 57</td><td> 840</td><td> 2</td><td> 100</td><td> 0</td>

<td> 10</td><td> 165</td><td> 2</td><td> 100</td><td> 0</td><td> 58</td><td> 885</td><td> 2</td><td> 100</td><td> 0</td>

<td> 1 1</td><td> 166</td><td> 2</td><td> 20</td><td> 80</td><td> 59</td><td> 886</td><td> 2</td><td> 78</td><td> 22</td>

<td> 12</td><td> 172</td><td> 2</td><td> 20</td><td> 80</td><td> 60</td><td> 892</td><td> 2</td><td> 78</td><td> 22</td>

<td> 13</td><td> 180</td><td> 2</td><td> 100</td><td> 0</td><td> 61</td><td> 900</td><td> 2</td><td> 100</td><td> 0</td>

<td> 14</td><td> 225</td><td> 2</td><td> 100</td><td> 0</td><td> 62</td><td> 945</td><td> 2</td><td> 100</td><td> 0</td>

<td> 15</td><td> 226</td><td> 2</td><td> 28</td><td> 72</td><td> 63</td><td> 946</td><td> 2</td><td> 80</td><td> 20</td>

<td> 16</td><td> 232</td><td> 2</td><td> 28</td><td> 72</td><td> 64</td><td> 952</td><td> 2</td><td> 80</td><td> 20</td>

<td> 17</td><td> 240</td><td> 2</td><td> 100</td><td> 0</td><td> 65</td><td> 960</td><td> 2</td><td> 100</td><td> 0</td>

<td> 18</td><td> 285</td><td> 2</td><td> 100</td><td> 0</td><td> 66</td><td> 1005</td><td> 2</td><td> 100</td><td> 0</td>

<td> 19</td><td> 286</td><td> 2</td><td> 34</td><td> 66</td><td> 67</td><td> 1006</td><td> 2</td><td> 82</td><td> 18</td>

<td> 20</td><td> 292</td><td> 2</td><td> 34</td><td> 66</td><td> 68</td><td> 1012</td><td> 2</td><td> 82</td><td> 18</td>

<td> 21</td><td> 300</td><td> 2</td><td> 100</td><td> 0</td><td> 69</td><td> 1020</td><td> 2</td><td> 100</td><td> 0</td>

<td> 22</td><td> 345</td><td> 2</td><td> 100</td><td> 0</td><td> 70</td><td> 1065</td><td> 2</td><td> 100</td><td> 0</td>

<td> 23</td><td> 346</td><td> 2</td><td> 42</td><td> 58</td><td> 71</td><td> 1066</td><td> 2</td><td> 84</td><td> 16</td>

<td> 24</td><td> 352</td><td> 2</td><td> 42</td><td> 58</td><td> 72</td><td> 1072</td><td> 2</td><td> 84</td><td> 16</td>

<td> 25</td><td> 360</td><td> 2</td><td> 100</td><td> 0</td><td> 73</td><td> 1080</td><td> 2</td><td> 100</td><td> 0</td>

<td> 26</td><td> 405</td><td> 2</td><td> 100</td><td> 0</td><td> 74</td><td> 1125</td><td> 2</td><td> 100</td><td> 0</td>

<td> 27</td><td> 406</td><td> 2</td><td> 50</td><td> 50</td><td> 75</td><td> 1126</td><td> 2</td><td> 86</td><td> 14</td>

<td> 28</td><td> 412</td><td> 2</td><td> 50</td><td> 50</td><td> 76</td><td> 1132</td><td> 2</td><td> 86</td><td> 14</td>

<td> 29</td><td> 420</td><td> 2</td><td> 100</td><td> 0</td><td> 77</td><td> 1140</td><td> 2</td><td> 100</td><td> 0</td>

<td> 30</td><td> 465</td><td> 2</td><td> 100</td><td> 0</td><td> 78</td><td> 1185</td><td> 2</td><td> 100</td><td> 0</td>

<td> 31</td><td> 466</td><td> 2</td><td> 54</td><td> 46</td><td> 79</td><td> 1186</td><td> 2</td><td> 88</td><td> 12</td>

<td> 32</td><td> 472</td><td> 2</td><td> 54</td><td> 46</td><td> 80</td><td> 1192</td><td> 2</td><td> 88</td><td> 12</td>

<td> 33</td><td> 480</td><td> 2</td><td> 100</td><td> 0</td><td> 81</td><td> 1200</td><td> 2</td><td> 100</td><td> 0</td>

<td> 34</td><td> 525</td><td> 2</td><td> 100</td><td> 0</td><td> 82</td><td> 1245</td><td> 2</td><td> 100</td><td> 0</td>

<td> 35</td><td> 526</td><td> 2</td><td> 58</td><td> 42</td><td> 83</td><td> 1246</td><td> 2</td><td> 90</td><td> 10</td>

<td> 36</td><td> 532</td><td> 2</td><td> 58</td><td> 42</td><td> 84</td><td> 1252</td><td> 2</td><td> 90</td><td> 10</td>

<td> 37</td><td> 540</td><td> 2</td><td> 100</td><td> 0</td><td> 85</td><td> 1260</td><td> 2</td><td> 100</td><td> 0</td>

<td> 38</td><td> 585</td><td> 2</td><td> 100</td><td> 0</td><td> 86</td><td> 1305</td><td> 2</td><td> 100</td><td> 0</td>

<td> 39</td><td> 586</td><td> 2</td><td> 62</td><td> 38</td><td> 87</td><td> 1306</td><td> 2</td><td> 95</td><td> 5</td>

<td> 40</td><td> 592</td><td> 2</td><td> 62</td><td> 38</td><td> 88</td><td> 1312</td><td> 2</td><td> 95</td><td> 5</td>

<td> 41</td><td> 600</td><td> 2</td><td> 100</td><td> 0</td><td> 89</td><td> 1319</td><td> 2</td><td> 100</td><td> 0</td>

<td> 42</td><td> 645</td><td> 2</td><td> 100</td><td> 0</td><td> 90</td><td> 1364</td><td> 2</td><td> 100</td><td> 0</td>

<td> 43</td><td> 646</td><td> 2</td><td> 66</td><td> 34</td><td></td><td></td><td></td><td></td><td></td>

<td> 44</td><td> 652</td><td> 2</td><td> 66</td><td> 34</td><td></td><td></td><td></td><td></td><td></td>

<td> 45</td><td> 660</td><td> 2</td><td> 100</td><td> 0</td><td></td><td></td><td></td><td></td><td></td>

<td> 46</td><td> 705</td><td> 2</td><td> 100</td><td> 0</td><td></td><td></td><td></td><td></td><td></td>

<td> 47</td><td> 706</td><td> 2</td><td> 70</td><td> 30</td><td></td><td></td><td></td><td></td><td></td>

<td> 48</td><td> 713</td><td> 2</td><td> 70</td><td> 30</td><td></td><td></td><td></td><td></td><td></td>

WO 94/18227

PCT/DK94/00054 ώ 1 3 3 ΰ û J 61

TABLE 3

<td> Step</td><td> Trne</td><td> Flow</td><td> %A</td><td></td><td> Step</td><td> Trne</td><td> Flow</td><td></td><td> %B</td>

<td> 1</td><td> 0,0</td><td> 1,0</td><td> 0,0</td><td> 100.0</td><td> 25.0</td><td> 420,5</td><td> 1.0</td><td> 60.0</td><td> 40,0</td>

<td> 2</td><td> 10,0</td><td> 1,0</td><td> 0,0</td><td> 100,0</td><td> 26,0</td><td> 430,0</td><td> 1,0</td><td> 60,0</td><td> 40,0</td>

<td> 3</td><td> 40,0</td><td> 1.0</td><td> 100,0</td><td> 0,0</td><td> 27,0</td><td> 460,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 4</td><td> 70,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 28,0</td><td> 490,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 5</td><td> 70,5</td><td> 1,0</td><td> 10,0</td><td> 90,0</td><td> 29,0</td><td> 490,5</td><td> 1,0</td><td> 70,0</td><td> 30,0</td>

<td> 6</td><td> 80,0</td><td> 1,0</td><td> 10,0</td><td> 90,0</td><td> 30,0</td><td> 500,0</td><td> 1,0</td><td> 70,0</td><td> 30,0</td>

<td> 7</td><td> 110,0</td><td> 1,0</td><td> 100,0</td><td> 0.0</td><td> 31,0</td><td> 530,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 8</td><td> 140,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 32,0</td><td> 560,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 9</td><td> 140,5</td><td> 1,0</td><td> 20,0</td><td> 80.0</td><td> 33,0</td><td> 560,5</td><td> 1.0</td><td> 80,0</td><td> 20,0</td>

<td> 10</td><td> 150,0</td><td> 1,0</td><td> 20,0</td><td> ao,o</td><td> 34,0</td><td> 570,0</td><td> l.o</td><td> 80,0</td><td> 20,0</td>

<td> 11</td><td> 180,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 35,0</td><td> 600,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 12</td><td> 210,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 36,0</td><td> 630,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 13</td><td> 210,5</td><td> 1,0</td><td> 30,0</td><td> 70,0</td><td> 37,0</td><td> 630,5</td><td> 1,0</td><td> 85,0</td><td> 15,0</td>

<td> 14</td><td> 220,0</td><td> 1,0</td><td> 30,0</td><td> 70,0</td><td> 38,0</td><td> 640,0</td><td> 1.0</td><td> 85,0</td><td> 15,0</td>

<td> 15</td><td> 250,0</td><td> 1.0</td><td> 100,0</td><td> 0,0</td><td> 39,0</td><td> 670,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 16</td><td> 280,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 40,0</td><td> 700,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 17</td><td> 280,5</td><td> 1,0</td><td> 40,0</td><td> 60,0</td><td> 41,0</td><td> 700,5</td><td> 1,0</td><td> 88,0</td><td> 12,0</td>

<td> 18</td><td> 290,0</td><td> 1,0</td><td> 40,0</td><td> 60,0</td><td> 42,0</td><td> 710,0</td><td> 1,0</td><td> 88,0</td><td> 12,0</td>

<td> 19</td><td> 320,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 43,0</td><td> 740,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 20</td><td> 350,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 44,0</td><td> 770,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 21</td><td> 350,5</td><td> 1,0</td><td> 50,0</td><td> 50,0</td><td> 45,0</td><td> 770,5</td><td> 1,0</td><td> 90,0</td><td> 10,0</td>

<td> 22</td><td> 360,0</td><td> 1.0</td><td> 50,0</td><td> 50,0</td><td> 46,0</td><td> 780,0</td><td> 1,0</td><td> 90,0</td><td> 10,0</td>

<td> 23</td><td> 390,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 47,0</td><td> 810,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td>

<td> 24</td><td> 420,0</td><td> 1,0</td><td> 100,0</td><td> 0,0</td><td> 48,0</td><td> 850,0</td><td> 1.0</td><td> 100,0</td><td> 0,0</td>

WO 94/18227

PCT/DK94/00054

2155335 ⁶²

TABLE 4

<td> ;ep</td><td> Tme</td><td> Flow</td><td> %A</td><td></td><td> Step</td><td> Tme</td><td> Flow</td><td> %A</td><td> %6</td>

<td> 1</td><td> 0</td><td> 2</td><td> 100</td><td> 0</td><td> 49</td><td> 720</td><td> 2</td><td> 100</td><td> 0</td>

<td> 2</td><td> 45</td><td> 2</td><td> 100</td><td> 0</td><td> 50</td><td> 765</td><td> 2</td><td> 100</td><td> 0</td>

<td> 3</td><td> 46</td><td> 2</td><td> 0</td><td> 100</td><td> 51</td><td> 766</td><td> 2</td><td> 48</td><td> 52</td>

<td> 4</td><td> 52</td><td> 2</td><td> 0</td><td> 100</td><td> 52</td><td> 772</td><td> 2</td><td> 48</td><td> 52</td>

<td> 5</td><td> 60</td><td> 2</td><td> 100</td><td> 0</td><td> 53</td><td> 780</td><td> 2</td><td> 100</td><td> 0</td>

<td> 6</td><td> 105</td><td> 2</td><td> 100</td><td> 0</td><td> 54</td><td> 825</td><td> 2</td><td> 100</td><td> 0</td>

<td> 7</td><td> 106</td><td> 2</td><td> 4</td><td> 96</td><td> 55</td><td> 826</td><td> 2</td><td> 52</td><td> 48</td>

<td> 6</td><td> 113</td><td> 2</td><td> 4</td><td> 96</td><td> 56</td><td> 832</td><td> 2</td><td> 52</td><td> 48</td>

<td> 9</td><td> 120</td><td> 2</td><td> 100</td><td> 0</td><td> 57</td><td> 840</td><td> 2</td><td> 100</td><td> 0</td>

<td> 10</td><td> 165</td><td> 2</td><td> 100</td><td> 0</td><td> 58</td><td> 885</td><td> 2</td><td> 100</td><td> 0</td>

<td> 1 1</td><td> 166</td><td> 2</td><td> 8</td><td> 92</td><td> 59</td><td> 886</td><td> 2</td><td> 56</td><td> 44</td>

<td> 12</td><td> 172</td><td> 2</td><td> 8</td><td> 92</td><td> 60</td><td> 892</td><td> 2</td><td> 56</td><td> 44</td>

<td> 13</td><td> 180</td><td> 2</td><td> 100</td><td> 0</td><td> 61</td><td> 900</td><td> 2</td><td> 100</td><td> 0</td>

<td> 14</td><td> 225</td><td> 2</td><td> 100</td><td> 0</td><td> 62</td><td> 945</td><td> 2</td><td> 100</td><td> 0</td>

<td> 1 5</td><td> 226</td><td> 2</td><td> 12</td><td> 88</td><td> 63</td><td> 946</td><td> 2</td><td> 60</td><td> 40</td>

<td> 16</td><td> 232</td><td> 2</td><td> 12</td><td> 88</td><td> 64</td><td> 952</td><td> 2</td><td> 60</td><td> 40</td>

<td> 17</td><td> 240</td><td> 2</td><td> 100</td><td> 0</td><td> 65</td><td> 960</td><td> 2</td><td> 100</td><td> 0</td>

<td> 18</td><td> 285</td><td> 2</td><td> 100</td><td> 0</td><td> 66</td><td> 1005</td><td> 2</td><td> 100</td><td> 0</td>

<td> 19</td><td> 286</td><td> 2</td><td> 16</td><td> 84</td><td> 67</td><td> 1006</td><td> 2</td><td> 64</td><td> 36</td>

<td> 20</td><td> 292</td><td> 2</td><td> 16</td><td> 84</td><td> 68</td><td> 1012</td><td> 2</td><td> 64</td><td> 36</td>

<td> 21</td><td> 300</td><td> 2</td><td> 100</td><td> 0</td><td> 69</td><td> 1020</td><td> 2</td><td> 100</td><td> 0</td>

<td> 22</td><td> 345</td><td> 2</td><td> 100</td><td> 0</td><td> 70</td><td> 1065</td><td> 2</td><td> 100</td><td> 0</td>

<td> 23</td><td> 346</td><td> 2</td><td> 20</td><td> 80</td><td> 71</td><td> 1066</td><td> 2</td><td> 68</td><td> 32</td>

<td> 24</td><td> 352</td><td> 2</td><td> 20</td><td> 80</td><td> 72</td><td> 1072</td><td> 2</td><td> 68</td><td> 32</td>

<td> 25</td><td> 360</td><td> 2</td><td> 100</td><td> 0</td><td> 73</td><td> 1080</td><td> 2</td><td> 100</td><td> 0</td>

<td> 26</td><td> 405</td><td> 2</td><td> 100</td><td> 0</td><td> 74</td><td> 1125</td><td> 2</td><td> 100</td><td> 0</td>

<td> 27</td><td> 406</td><td> 2</td><td> 24</td><td> 76</td><td> 75</td><td> 1126</td><td> 2</td><td> 70</td><td> 30</td>

<td> 28</td><td> 412</td><td> 2</td><td> 24</td><td> 76</td><td> 76</td><td> 1132</td><td> 2</td><td> 70</td><td> 30</td>

<td> 29</td><td> 420</td><td> 2</td><td> 100</td><td> 0</td><td> 77</td><td> 1140</td><td> 2</td><td> 100</td><td> 0</td>

<td> 30</td><td> 465</td><td> 2</td><td> 100</td><td> 0</td><td> 78</td><td> 1185</td><td> 2</td><td> 100</td><td> 0</td>

<td> 31</td><td> 466</td><td> 2</td><td> 28</td><td> 72</td><td> 79</td><td> 1186</td><td> 2</td><td> 72</td><td> 28</td>

<td> 32</td><td> 472</td><td> 2</td><td> 28</td><td> 72</td><td> 80</td><td> 1192</td><td> 2</td><td> 72</td><td> 28</td>

<td> 33</td><td> 480</td><td> 2</td><td> 1 00</td><td> 0</td><td> 81</td><td> 1200</td><td> 2</td><td> 100</td><td> 0</td>

<td> 34</td><td> 525</td><td> 2</td><td> 100</td><td> 0</td><td> 82</td><td> 1245</td><td> 2</td><td> 100</td><td> 0</td>

<td> 35</td><td> 526</td><td> 2</td><td> 32</td><td> 68</td><td> 83</td><td> 1246</td><td> 2</td><td> 75</td><td> 25</td>

<td> 36</td><td> 532</td><td> 2</td><td> 32</td><td> 68</td><td> 84</td><td> 1252</td><td> 2</td><td> 75</td><td> 25</td>

<td> 37</td><td> 540</td><td> 2</td><td> 100</td><td> 0</td><td> 85</td><td> 1260</td><td> 2</td><td> 100</td><td> 0</td>

<td> 38</td><td> 585</td><td> 2</td><td> 100</td><td> 0</td><td> 86</td><td> 1305</td><td> 2</td><td> 100</td><td> 0</td>

<td> 39</td><td> 586</td><td> 2</td><td> 36</td><td> 64</td><td> 87</td><td> 1306</td><td> 2</td><td> 80</td><td> 20</td>

<td> 40</td><td> 592</td><td> 2</td><td> 36</td><td> 64</td><td> 88</td><td> 1312</td><td> 2</td><td> 80</td><td> 20</td>

<td> 41</td><td> 600</td><td> 2</td><td> 100</td><td> 0</td><td> 89</td><td> 1319</td><td> 2</td><td> 100</td><td> 0</td>

<td> 42</td><td> 645</td><td> 2</td><td> 100</td><td> 0</td><td> 90</td><td> 1364</td><td> 2</td><td> 100</td><td> 0</td>

<td> 43</td><td> 646</td><td> 2</td><td> 40</td><td> 60</td><td> 91</td><td> 1365</td><td> 2</td><td> 85</td><td> 1 5</td>

<td> 44</td><td> 652</td><td> 2</td><td> 40</td><td> 60</td><td> 92</td><td> 1371</td><td> 2</td><td> 85</td><td> 1 5</td>

<td> 45</td><td> 660</td><td> 2</td><td> 100</td><td> 0</td><td> 93</td><td> 1378</td><td> 2</td><td> 100</td><td> 0</td>

<td> 46</td><td> 705</td><td> 2</td><td> 100</td><td> 0</td><td> 94</td><td> 1423</td><td> 2</td><td> 100</td><td> 0</td>

<td> 47</td><td> 706</td><td> 2</td><td> 44</td><td> S6</td><td></td><td></td><td></td><td></td><td></td>

<td> 48</td><td> 71 3</td><td> 2</td><td> 44</td><td> 56</td><td></td><td></td><td></td><td></td><td></td>

π iJDJûD PCT/DK94/00054

TABLE 5

<td> Step</td><td> Trne</td><td> Flow</td><td> %A</td><td></td><td> Step</td><td> Trne</td><td> Flow</td><td> %A</td><td> %B</td>

<td> 1</td><td> 0</td><td> 2</td><td> 100</td><td> 0</td><td> 49</td><td> 720</td><td> 2</td><td> 100</td><td> 0</td>

<td> 2</td><td> 45</td><td> 2</td><td> 100</td><td> 0</td><td> 50</td><td> 765</td><td> 2</td><td> 100</td><td> 0</td>

<td> 3</td><td> 46</td><td> 2</td><td> 0</td><td> 100</td><td> 51</td><td> 766</td><td> 2</td><td> 52</td><td> 48</td>

<td> 4</td><td> 52</td><td> 2</td><td> 0</td><td> 100</td><td> 52</td><td> 772</td><td> 2</td><td> 52</td><td> 48</td>

<td> 5</td><td> 60</td><td> 2</td><td> 100</td><td> 0</td><td> 53</td><td> 780</td><td> 2</td><td> 100</td><td> 0</td>

<td> 6</td><td> 105</td><td> 2</td><td> 100</td><td> 0</td><td> 54</td><td> 825</td><td> 2</td><td> 100</td><td> 0</td>

<td> 7</td><td> 106</td><td> 2</td><td> 13</td><td> 87</td><td> 55</td><td> 826</td><td> 2</td><td> 54</td><td> 46</td>

<td> 8</td><td> 113</td><td> 2</td><td> 13</td><td> 87</td><td> 56</td><td> 832</td><td> 2</td><td> 54</td><td> 46</td>

<td> 9</td><td> 120</td><td> 2</td><td> 100</td><td> 0</td><td> 57</td><td> 840</td><td> 2</td><td> 100</td><td> 0</td>

<td> 10</td><td> 165</td><td> 2</td><td> 100</td><td> 0</td><td> 58</td><td> 88S</td><td> 2</td><td> 100</td><td> 0</td>

<td> η</td><td> 166</td><td> 2</td><td> 25</td><td> 75</td><td> 59</td><td> 886</td><td> 2</td><td> 56</td><td> 44</td>

<td> 12</td><td> 172</td><td> 2</td><td> 25</td><td> 75</td><td> 60</td><td> 892</td><td> 2</td><td> 56</td><td> 44</td>

<td> 13</td><td> 180</td><td> 2</td><td> 100</td><td> 0</td><td> 61</td><td> 900</td><td> 2</td><td> 100</td><td> 0</td>

<td> 14</td><td> 225</td><td> 2</td><td> 100</td><td> 0</td><td> 62</td><td> 945</td><td> 2</td><td> 100</td><td> 0</td>

<td> 15</td><td> 226</td><td> 2</td><td> 29</td><td> 71</td><td> 63</td><td> 946</td><td> 2</td><td> 58</td><td> 42</td>

<td> 16</td><td> 232</td><td> 2</td><td> 29</td><td> 71</td><td> 64</td><td> 952</td><td> 2</td><td> 58</td><td> 42</td>

<td> 17</td><td> 240</td><td> 2</td><td> 100</td><td> 0</td><td> 65</td><td> 960</td><td> 2</td><td> 100</td><td> 0</td>

<td> 18</td><td> 285</td><td> 2</td><td> 100</td><td> 0</td><td> 66</td><td> 1005</td><td> 2</td><td> 100</td><td> 0</td>

<td> 19</td><td> 286</td><td> 2</td><td> 34</td><td> 66</td><td> 67</td><td> 1006</td><td> 2</td><td> 60</td><td> 40</td>

<td> 20</td><td> 292</td><td> 2</td><td> 34</td><td> 66</td><td> 68</td><td> 1012</td><td> 2</td><td> 60</td><td> 40</td>

<td> 21</td><td> 300</td><td> 2</td><td> 100</td><td> 0</td><td> 69</td><td> 1020</td><td> 2</td><td> 100</td><td> 0</td>

<td> 22</td><td> 345</td><td> 2</td><td> 100</td><td> 0</td><td> 70</td><td> 1065</td><td> 2</td><td> 100</td><td> 0</td>

<td> 23</td><td> 346</td><td> 2</td><td> 38</td><td> 62</td><td> 71</td><td> 1066</td><td> 2</td><td> 62</td><td> 33</td>

<td> 24</td><td> 352</td><td> 2</td><td> 38</td><td> 62</td><td> 72</td><td> 1072</td><td> 2</td><td> 62</td><td> 38</td>

<td> 25</td><td> 360</td><td> 2</td><td> 100</td><td> 0</td><td> 73</td><td> 1080</td><td> 2</td><td> 100</td><td> 0</td>

<td> 26</td><td> 405</td><td> 2</td><td> 100</td><td> 0</td><td> 74</td><td> 1125</td><td> 2</td><td> 100</td><td> 0</td>

<td> 27</td><td> 406</td><td> 2</td><td> 40</td><td> 60</td><td> 75</td><td> 1126</td><td> 2</td><td> 66</td><td> 34</td>

<td> 28</td><td> 412</td><td> 2</td><td> 40</td><td> 60</td><td> 76</td><td> 1132</td><td> 2</td><td> 66</td><td> 34</td>

<td> 29</td><td> 420</td><td> 2</td><td> 100</td><td> 0</td><td> 77</td><td> 1140</td><td> 2</td><td> 100</td><td> 0</td>

<td> 30</td><td> 465</td><td> 2</td><td> 100</td><td> 0</td><td> 78</td><td> 1185</td><td> 2</td><td> 100</td><td> 0</td>

<td> 31</td><td> 466</td><td> 2</td><td> 42</td><td> 58</td><td> 79</td><td> 1186</td><td> 2</td><td> 70</td><td> 30</td>

<td> 32</td><td> 472</td><td> 2</td><td> 42</td><td> 58</td><td> 80</td><td> 1192</td><td> 2</td><td> 70</td><td> 30</td>

<td> 33</td><td> 480</td><td> 2</td><td> 100</td><td> 0</td><td> 81</td><td> 1200</td><td> 2</td><td> 100</td><td> 0</td>

<td> 34</td><td> 525</td><td> 2</td><td> 100</td><td> 0</td><td> 82</td><td> 1245</td><td> 2</td><td> 100</td><td> 0</td>

<td> 35</td><td> 526</td><td> 2</td><td> 44</td><td> 56</td><td> 83</td><td> 1246</td><td> 2</td><td> 74</td><td> 26</td>

<td> 36</td><td> 532</td><td> 2</td><td> 44</td><td> 56</td><td> 84</td><td> 1252</td><td> 2</td><td> 74</td><td> 26</td>

<td> 37</td><td> 540</td><td> 2</td><td> 100</td><td> 0</td><td> 85</td><td> 1260</td><td> 2</td><td> 100</td><td> 0</td>

<td> 38</td><td> 585</td><td> 2</td><td> 100</td><td> 0</td><td> 86</td><td> 1305</td><td> 2</td><td> 100</td><td> 0</td>

<td> 39</td><td> 586</td><td> 2</td><td> 46</td><td> 54</td><td> 87</td><td> 1306</td><td> 2</td><td> 78</td><td> 22</td>

<td> 40</td><td> 592</td><td> 2</td><td> 46</td><td> 54</td><td> 88</td><td> 1312</td><td> 2</td><td> 78</td><td> 22</td>

<td> 41</td><td> 600</td><td> 2</td><td> 100</td><td> 0</td><td> 89</td><td> 1319</td><td> 2</td><td> 100</td><td> 0</td>

<td> 42</td><td> 645</td><td> 2</td><td> 100</td><td> 0</td><td> 90</td><td> 1364</td><td> 2</td><td> 100</td><td> 0</td>

<td> 43</td><td> 646</td><td> 2</td><td> 48</td><td> 52</td><td> 91</td><td> 1365</td><td> 2</td><td> 82</td><td> 18</td>

<td> 44</td><td> 652</td><td> 2</td><td> 48</td><td> 52</td><td> 92</td><td> 1371</td><td> 2</td><td> 82</td><td> 18</td>

<td> 45</td><td> 660</td><td> 2</td><td> 100</td><td> 0</td><td> 93</td><td> 1378</td><td> 2</td><td> 100</td><td> 0</td>

<td> 46</td><td> 705</td><td> 2</td><td> 100</td><td> 0</td><td> 94</td><td> 1423</td><td> 2</td><td> 100</td><td> 0</td>

<td> 47</td><td> 706</td><td> 2</td><td> 50</td><td> 50</td><td></td><td></td><td></td><td></td><td></td>

<td> 48</td><td> 71 3</td><td> 2</td><td> 50</td><td> 50</td><td></td><td></td><td></td><td></td><td></td>

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EXAMPLE 1

Production and Folding of Human and Murine β₂-microglobulin

This example describes the production in E. coli of both human β₂-microglobulin and murine β₂-microglobulin as FX_acleavable fusion proteins, and the purification of the recombinant human and murine β₂-microglobulin after FX_a cleavage.

Plasmid clones containing the full length cDNAs encoding the human and the murine β₂-microglobulin proteins (generouslyprovided by Dr. David N. Garboczi to Dr. Soren Buus) were used as templates in a Polymerase Chain Reaction (PCR) (Saiki et al., 1988) designed to produce cDNA fragments corresponding to the mature human (corresponding to amino acid residue Ile₁ to Met₉₉) and the mature murine (corresponding to amino acid residue Ile^^ to Met₉₉) β₂-microglobulin proteins, by use of the primers SEQ ID NO: 3 and SEQ ID NO: 4 (for the human Ê₂-microglobulin) and SEQ ID NO: 5 and SEQ ID NO: 6 (for the murine S₂-microglobulin) · ^The amplified coding reading frames were at their 5'-ends, via the PCR-reaction, linked to nucleotide sequences, included in SEQ ID NO: 3 and 5, encoding the amino acid sequence SEQ ID NO: 37, which constitute a cleavage site for the bovine restriction protease FX_a(Nagai and Thogersen, 1987). The amplified DNA fragments were subcloned into the E. coli expression vector pT₇H₆ (ChriThe construction of the resulting expressing human β₂-microglobulin) and pT₇HgFX-m^₂^m (expressing murine β₂-microglobulin) is outlined in fig. 2 and in fig. 3 is shown the amino acid sequences of the expressed proteins (in SEQ ID NO: 49 (human) and SEQ ID NO: 50 (murine) are shown the amino acid sequences encoded by the full length reading frames).

stensen et al., 1991) plasmids pT₇HgFX-hjS₂ni

Human and murine β₂-microglobulin were produced by growing and expressing the plasmids pT₇H₆FX-h0₂m and -m/3₂m in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986. ExpoWO 94/18227

PCTfDK$4/00054 nentially growing cultures at 37°C were at OD₆₀₀ 0.8 infected with bacteriophage XCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2-,-5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chlo10 ride, 50 mM Tris-HCl pH 8 and 0.1 M,dithioerythrifel., FollowΎΜ ing gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 1 M NaCl, 50 mM Tris-HCl pH 8, 10 mM 2-mercaptoethanol and 3 mM methionine the crude protein preparation was applied to Ni²⁺ activated NTA-agarose columns for purification (Hochuli et al., 1988.) of the fusion proteins, MGSHHHHHHGSIEGR-human and murine β₂-microglobulin (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) respectively and subsequently to undergo the cyclic folding procedure.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

Ni²⁺ activated NTA-agarose matrix (Ni²⁺NTA-agarose) is commercially available from Diagen GmbH, Germany. During the course of this work it was found, however, that this commercial product did not perform as well as expected. Our obser25 vations were, that the commercial Ni²⁺NTA-agarose matrix was easily blocked when applying the denatured and reduced total protein extract, that the capacity for fusion protein was lower than expected, and that the matrix could only be regenerated successfully a few times over.

In order to improve the performance of the Ni²⁺NTA-agarose it was decided to perform a carbodiimide coupling of the N-(5amino-1-carboxypentyl) iminodiacetic acid metal ligand (synthesis route as described by Dôbeli &amp; Hochuli (EPO 0253 303))

TM to a more rigid agarose matrix (i.e. Sepharose CL-6B, Pharma35 cia, Sweden):

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g. of N-(5-amino-l-carboxypentyl)iminodiacetic acid from the synthesis procedure in 50 ml was adjusted to pH 10 by addition of 29 g. of Na₂C0₃(10 H₂O) and added to a stirred suspension of activated Sepharose CL-6B in 1 M Na₂CO₃. Reac5 tion was allowed overnight.

The Sepharose CL-6B (initially 100 ml. suspension) was activated after removal of water by acetone with 7 g. of 1,1'carbonyldiimidazol under stirring for 15 to 30 min. Upon activation the Sepharose CL-6B was washed with acetone folio lowed by water and 1 M Na₂CO₃. The NTA-agarose matrix was loaded into a column and charged” with Ni²⁺ by slowly passing through 5 column volumes of a 10% NiS0₄ solution. The amount of Ni²⁺ on the NTA-agarose matrix, prepared by this procedure, has been determined to 14 mmoles per ml matrix.

The Ni²⁺NTA-agarose matrix was packed in a standard class column for liquid chromatography (internal diameter: 2.6 cm) to a volume of 40 ml. After charging the Ni²⁺NTA-agarose column was washed with two column volumes of water, one column volume of 1 M Tris-HCl pH 8 and two column volumes of loading buffer before application of the crude protein extract.

Upon application of the crude protein extracts on the Ni²⁺NTA-agarose column, the fusion proteins, MGSHHHHHHGSIEGRh/3₂m and MGSHHHHHHGSIEGR-m/3₂m (wherein MGSHHHHHHGSIEGR is SEQ

ID NO: 48) respectively, were purified from the majority of coli and λ phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, mM Tris-HCl, 10 mM 2-mercaptoethanol, and 3 mM methionine until the optical density (OD) at 280 nm of the column elu30 ates were stable.

The fusion proteins were refolded on the Ni²⁺NTA-agarose column using a gradient manager profile as described in table 1 and 0.5 M NaCl, 50 mM Tris-HCl pH 8, and 1.2 mM/0.4 mM reduced/oxidized gluthatione^as buffer A and 8 M urea, 0.5 M

NaCl, 50 mM Tris-HCl pH 8,. 3 mM methionine, and 6 mM reduced

WO. 94/18227

PCT/DK94/00054 gluthatione as buffer B. The reduced/oxidized gluthatione solution was freshly prepared as a 200 times stock solution by addition of 9.9 M K₂O₂ to a stirred solution of 0.2 M reduced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure the h/S₂m and m/?₂m fusion proteins were eluted from the Ni²⁺NTA-agarose columns with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, 20 mM EDTA pH 8.

Fusion protein that were aggregated and precipitated on the 10 Ni²⁺NTA-agarose columns were eluted in buffer B.

Approximately 75% of the fusion protein material was eluted by non-denaturing elution buffer (see Fig. 16, lanes 2 and 3) .

As judged by non-reducing SDS-PAGE analysis approximately 70 15 % of the soluble h0₂m fusion protein material (corresponding to 40 mg of h/S₂^m fusion protein) appeared monomeric (see Fig. 15, lanes 5 and 3) whereas 25 % of the m0₂m fusion protein appeared monomeric (corresponding to 20 mg of m/?₂m fusion protein). The overall efficiency of the folding procedure are therefore approximately 50 % for the h/3₂m fusion protein and less than 20% for the m/î₂m fusion protein.

Monomeric hf?₂m and m/3₂m fusion proteins were' purified from dimer and higher order multimers by ion exchange chromatog-TM raphy on S-Sepharose (Pharmacia, Sweden): The fusion proteins 25 eluted by the non denaturing elution buffer (approximately 70 % of the fusion protein material) was gelfiltrated into a buffer containing 5 mM NaCl and 5 mM Tris-HCl pH 8 on Sepha-ΓΜ dex G-25 and diluted 1:1 with water before applied onto the

-1M ,

S-Sepharose ion exchange columns. Fusion proteins were eluted over 5 column volumes with a liner gradient from 2.5 mM NaCl, 2.5 mM Tris-Hcl pH 8 to 100 mM NaCl, 25 mM Tris-Hcl pH 8. The monomeric h0₂m as well as m$₂m fusion proteins eluted in the very beginning of the gradient, whereas dimers and higher order multimers eluted later. Fractions containing the

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<img file="CA2155335C_D0010.tif" />

monomeric fusion proteins were diluted with water and reloaded to the S-Sepharose columns and one-step eluted in 1 M NaCl, 50 mM Tris-HCl pH 8.

The monomeric rusion proteins were cleaved with the restriction protease FX_a overnight at room temperature in a weight to weight ratio of approximately 200 to one.

After cleavage the recombinant h/?₂m and mj3₂m proteins were purified from the N-terminal fusion tail, liberated from the cleaved fusion protein and FX_a by ion exchange chromatography on Q-Sepharose columns (Pharmacia, Sweden): Upon gelfiltration on Sephadex G-25 into 5 mM NaCl, 5 mM Tris-HCl pH 8 and 1:1 dilution with water, recombinant h/3₂m and m/3₂*n were eluted in a linear gradient (over 5 column volumes) from 2.5 mM NaCl, 2.5 mM Tris-HCl pH 8 to 100 mM NaCl, 25 mM Tris-HCl pH 8. Fractions containing the cleaved recombinant proteins were diluted with water and reloaded to the Q-Sepharose columns and one-step eluted in 1 M NaCl, 50 mM Tris-HCl pH 8. Recombinant h/3₂m and m/J₂m proteins were gelfiltrated into freshly prepared 20 mM NH₄HCO₃ and lyophilized twice.

SDS-PAGE analysis of the production of recombinant human β₂microglobulin is presented in fig. 15.

The yield of fully processed recombinant human 0₂-microglobulin produced by this procedure was 30 mg.

The yield of fully processed recombinant murine β₂-microglobulin produced by this procedure was 10 mg.

Comparison of recombinant human with purified natural human β₂-microglobulin 3₂-microglobulin was kindly carried out by Dr. Soren Buus in two different assays:

1. It was found that Recombinant human β₂-microglobulin and natural human β₂-microglobulin reacted with both a monoclonal- and a monospecific antibody with identical affinity.

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2. Recombinant human β₂-microglobulin and natural human β₂microglobulin were in an binding inhibition experiment using radiolabelled ligands found to bind natural affinity purified heavy chain class I K^d molecules with an identical affinity.

Recombinant murine /3₂-microglobulin was found to bind natural class I heavy chain molecules with an affinity 5 times lower than the human β₂-microglobulin. This result is in good agreement with previous results from the literature using natural material.

EXAMPLE 2

Production and folding of Hunan Growth Hormone (Somatotropin)'

This example describes the production in E. coli of human growth hormone (hGH) as a FX_a cleavable fusion protein, and the purification of the recombinant hGH after FX_a cleavage.

A plasmid clone containing the cDNA encoding the hGH (generously provided by Dr. Henrik Dalboge (Dalboge et al., 1987) were used as template in a Polymerase Chain Reaction (PCR) (Saiki et al., 1988), using the primers SEQ ID NO: 7 and SEQ ID NO: 8, designed to produce a cDNA fragment corresponding to the mature hGH (corresponding to amino acid residue Phe₁to Phe₁₉₁) protein. The amplified coding reading frame was at the 5'-end, via the PCR-reaction, linked to a nucleotide sequence, included in SEQ ID NO: 7, encoding the amino acid sequence SEQ ID NO: 37 which constitute a cleavage site for the bovine restriction protease FX_a (Nagai and Thogersen,

1987). The amplified DNA fragment was subcloned into the E. coli expression vector pT₇H₆ (Christensen et al., 1991) . The construction of the resulting plasmid pT₇H₆FX-hGH (expressing human Growth Hormone) is outlined in fig. 4 and in fig. 5 is shown the amino acid sequence of the expressed protein (in

SEQ ID NO: 51 is shown the amino acid sequence encoded by the full length reading frame).

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PCT/DK94/00Û54

Recombinant human Growth Hormone was produced by growing and expressing the plasmid pT₇H₆FX-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986. Exponentially growing cultures at 37°C were at OD₆₀₀ 0.8 infected with bacteriophage ÂCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base).

Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Follow15 ing gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 1 M NaCl, 50 mM Tris-HCl pH 8, 5 mM 2-mercaptoethanol and 1 mM methionine the crude protein preparation was applied to a Ni²⁺ activated NTA-agarose column (Ni²⁺NTAagarose) for purification (Hochuli et al., 1988) of the fusion protein, MGSHHHHHHGSIEGR-hGH (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) and subsequently to undergo the cyclic folding procedure.

Preparation and charging of the Ni²⁺NTA-agarose column is described under Example 1.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

Upon application of the crude protein extract on the Ni²⁺NTAagarose column, the fusion protein, MGSHHHHHHGSIEGR-hGH (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) was purified from the majority of coli and λ phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl, 5 mM 2-mercaptoethanol, and 1 mM methionine until the optical density (OD) at 280 nm of the eluate was stable.

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The fusion protein was refolded on the Ni²⁺NTA-agarose column using a gradient manager profile as described in table 2 and 0.5 M NaCl, 50 xnM Tris-HCl pH 8, and 1.0 mM/0.1 mM reduced/oxidized gluthatione as buffer A and 8 M urea, 0.5 M 5 NaCl, 50 mM Tris-HCl pH 8, 1 mM methionine, and 5 mM reduced gluthatione as buffer B. The reduced/oxidized gluthatione solution was freshly prepared as a 200 times stock solution by addition of 9.9 M H₂O₂ to a stirred solution of 0.2 M reduced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure the hGH fusion protein was eluted from the Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, 20 mM EDTA pH 8. Fusion protein that were aggregated and precipitated on the Ni²⁺NTA-agarose column was eluted in buffer B.

Approximately 80% of the fusion protein material was eluted by the non denaturing elution buffer (see Fig. 16, lanes 2 and 3). As judged by non-reducing SDS-PAGE analysis 90 % of the soluble fusion protein material (corresponding to approximately 70 mg of fusion protein) appeared monomeric (see Fig.

16, lane 2) yielding an overall efficiency of the folding procedure of approximately 70 %.

Monomeric hGH fusion protein was purified from dimer and higher order multimers by ion exchange chromatography on QSepharose (Pharmacia, Sweden): After gelfiltration into a buffer containing 25 mM NaCl and 25 mM Tris-HCl pH 8 on

Sephadex G-25 the fusion protein material, eluted by the nondenaturing buffer, was applied onto a Q-Sepharose ion exchange column. Fusion protein were eluted over 5 column volumes with a linear gradient from 25 mM NaCl, 25 mM Tris30 HC1 pH 8 to 200 mM NaCl, 50 mM Tris-HCl pH 8. The monomeric hGH fusion protein eluted in the beginning of the gradient, whereas dimers and higher order multimers eluted later. Fractions containing the pure monomeric fusion protein was added NiSO₄ and iminodiacetic acid (IDA, adjusted pH 8 with

NaOH) to 1 mM and cleaved with the restriction protease FX_a

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<img file="CA2155335C_D0012.tif" />

for 5 hours at 37°C in a weight to weight ratio of approximately 100 to one. FX_a is inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.

After cleavage the recombinant hGH protein was isolated from uncleaved fusion protein and the liberated fusion tail, upon gelfiltration on Sephadex G-25 into 8 M Urea, 50 mM Tris-HCl pH 8, to remove Ni²⁺IDA and Benzamidine, by passage through a small Ni²⁺NTA-agarose column followed inline by a small Nd³⁺NTA agarose column and subsequently a non Ni²⁺activated NTA-agarose column to ensure complete removal of FXa and of Ni²⁺ and Nd³⁺, respectively. Recombinant hGH was purified from a minor fraction of recombinant breakdown product by ion exchange chromatography on Q-Sepharose: hGH was eluted in a linear gradient (over 5 column volumes) from 8 M Urea, 50 mM Tris-HCl pH 8 to 8 M Urea, 250 mM NaCI, 25 mM Tris-HCl pH 8. Fractions containing the cleaved purified recombinant protein was gelfiltrated into freshly prepared 20 mM NH4HCO₃ and lyophilized twice.

SDS-PAGE analysis of the production and folding of recombinant human growth hormone is presented in fig. 16.

The yield of fully processed recombinant human growth hormone produced by this procedure was 10 mg.

The recombinant human growth hormone produced by this procedure co-migrated both in reducing and non-reducing SDS-PAGE and in non-denaturing PAGE analysis with biologically active recombinant human growth hormone generously provided by NovoNordisk A/S.

EXAMPLE 3

Production and folding of human 012MRAP

The plasmid used for expression in E. coli BL21 cells of the human -Macroglobulin Receptor Associated Protein (œ₂MRAP), jyO 94/18227

PCT/DK94/00054 ' pT7H6FX-ûi₂MRAP and the conditions used for production of the fusion protein has previously been described by us in, Nykjær et al., J. Biol. Chem. 267: 14543-14546, 1992. The primers SEQ ID NO: 9 and SEQ ID NO: 10 were used in the PCR employed for multiplying the a₂MRAP encoding DNA.

Crude protein extract precipitated from the phenol phase of the protein extraction of cells from 2 litres of culture of MGSHHHHHHGSIEGR-a₂MRAP (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) expressing E. coli BL21 cells was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, Sweden) into 8 M Urea, 0.5 M NaCl, 50 mM Tris-HCl pH 8, and 1 mM methionine the crude protein preparation was applied to a Ni²⁺activated NTA-agarose matrix (Ni²⁺NTA-agarose) for purification (Hochuli et al., 1988) of the fusion protein, MGSHHHHHHGSIEGR-a₂MRAP (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) and subsequently to undergo the cyclic folding process.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

Preparation and charging of the Ni²⁺NTA-agarose column is described under Example 1.

Upon application of the crude protein extract on the Ni²⁺NTAagarose column, the fusion protein, MGSHHHHHHGSIEGR-a₂MRAP (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) was purified from the majority of coli and λ phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl, and 1 mM methionine until the optical density (OD) at 280 nm of the eluate was stable.

0 The fusion protein was refolded on the Ni²⁺NTA-agarose column using a gradient manager profile as described in table 3 and 0.5 M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl₂ and 1 mM 2-mercaptoethanol as buffer A and 6 M guanidinium chloride, 50 mM

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Tris-HCl pH 8, 2 mM CaCl₂ and 1 mM 2-mercaptoethanol as buffer B.

After completion of the cyclic folding procedure the a₂MRAP fusion protein was eluted from the Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, 20 mM EDTA pH 8.

Virtually no fusion protein was found to be aggregated or precipitated on the Ni²⁺NTA-agarose column. The estimated yield of a₂MRAP fusion protein was 60 mg and the efficiency of the folding procedure close to 95%.

The fusion protein MGSHHHHHHGSIEGR-œ₂MRAP (wherein

MGSHHHHHHGSIEGR is SEQ ID NO : 48) was cleaved with the bovine restriction protease FX_a overnight at room temperature in a weight to weight ratio of 200:1 in the elution buffer. Upon gelfiltration on Sephadex G-25 into 100 mM NaCl, 25 mM TrisHCl pH 8, the protein solution was passed through a Ni²⁺NTAagarose column thereby removing uncleaved fusion protein and the liberated fusion N-terminal tail originating from cleaved fusion proteins. Finally the protein solution was diluted 1:4 with water and the a₂MRAP protein purified from FX_a by ion exchange chromatography on Q-Sepharose (Pharmacia, Sweden). The Q-Sepharose column was eluted with a linear gradient over 6 column volumes from 25 mM NaCl, 25 mM Tris-HCl pH 8 to 250 mM NaCl, 25 mM Tris-HCl pH 8. The œ₂MRAP protein eluted in the very beginning of the linear gradient whereas FX_a eluted later.

The yield of œ₂MRAP protein produced and refolded by this procedure was 40 mg.

The ligand binding characteristics (i.e. binding to the a₂Macroglobulin Receptor and interference with the binding of human Urokinase Plasminogen Activator - Plasminogen Activator Inhibitor type-I complex to the œ₂-M Receptor) has, according jyo 94/18227

PCT/DK94/00054 to Dr. Nykjær, been found identical to the ligand binding characteristics of the purified natural protein.

EXAMPLE 4

Production and folding of domains and domain-clusters from 5 the oî₂~^m Receptor

The human a₂-Macroglobulin Receptor/Low Density Lipoprotein Receptor-Related Protein (ûî₂MR) is a 600 kDa endocytotic membrane receptor. a₂-MR is synthesized as a 4524 amino acid single chain precursor protein. The precursor is processed into a 85 kDa transmembrane 0-chain and a 500 kDa a-chain, non-covalently bound to the extracellular domain of the βchain. The a₂-MR is known to bind Ca²⁺ in a structure dependent manner (i.e. the reduced protein does not bind Ca²⁺) and is believed to be multifunctional in the sense that a₂-MR binds ligands of different classes.

The entire amino acid sequence of the a-chain can be represented by clusters of three types of repeats also found in other membrane bound receptors and in various plasma proteins :

A: This type of repeat span approximately 40 amino acid residues and is characterised by the sequential appearance of the six cysteinyl residues contained in the repeat. Some authors has named this repeat complement-type domain.

B: This type of repeat also span approximately 40 amino acid residues and is characterised by the sequential appearance of the six cysteinyl residues contained in the repeat. In the literature this repeat has been named EGF-type domains.

C: This type of repeat span approximately 55 amino acid residues and is characterised by the presence of the con30 sensus sequence SEQ ID NO: 39.

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215533576

This example describes the production in E. coli of a number of domains and domain-clusters derived from the œ₂-MR protein as FX_a cleavable fusion proteins and the purification, in vitro folding, and the FX_a cleavage and processing of these recombinant proteins.

A plasmid clone containing the full length cDNA encoding the human a₂-MR protein (generously provided by Dr. Joachim Herz; Herz et al._t EMBO J., 7: 4119-4127, 1988) were used as template in a series of Polymerase Chain Reactions (PCR) designed to produce cDNA fragments corresponding to a number of polypeptides representing domains and domain-clusters derived from the œ₂-MR protein:

#1: Contains two domains of the A-type, corresponding to amino acid residue 20 to 109 in the a₂-MR protein. The primers SEQ ID NO: 11 and SEQ ID NO: 12 were used in the PCR.

#2: Contains two domains of the A-type followed by two type-B domains, corresponding to amino acid residue 20 to 190 in the’ a₂-MR protein. The primers SEQ ID NO: 11 and SEQ ID NO: 13 were used in the PCR.

#3: Identical to #2 followed by a region containing YWTD repeats, corresponding to amino acid residue 20 to 521. The primers SEQ ID NO: 11 and SEQ ID NO: 14 were used in the PCR.

#4: Contains one type-B domain, followed by 8 type-A domains and finally two type-B domains, corresponding to amino acid residue 803 to 1265 in the a₂-MR protein. The primers SEQ ID NO: 15 and SEQ ID NO: 16 were used in the PCR.

#5: Contains only the 8 type-A domains also present in #4, corresponding to amino acid residue 849 to 1184 in the a₂-MR protein. The primers SEQ ID NO: 17 and SEQ ID NO: 18 were used in the PCR.

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PCT/DK94/00054 #6: Contains the two C-terminal type-B domains from #4, followed by 8 YWTD repeats and one type-B domain, corresponding to amino acid residue 1184 to 1582 in the a₂-MR protein. The primers SEQ ID NO: 19 and SEQ ID NO: 20 were used in the

PCR.

#7: Contains the whole region included in constructs #4 to #6, corresponding to amino acid residue 803 to 1582 in the a₂-MR protein. The primers SEQ ID NO: 15 and SEQ ID NO: 20 were used in the PCR.

#8: Contains 10 type-A domains, corresponding to amino acid residue 2520 to 2941 in the œ₂-MR protein. The primers SEQ ID NO: 21 and SEQ ID NO: 22 were used in the PCR.

#9: Contains 11 type-A domains, corresponding to amino acid residue 3331 to 3778 in the a₂-MR protein. The primers SEQ ID

NO: 23 and SEQ ID NO: 24 were used in the PCR.

The amplified nucleotide sequences encoding the domains and domain-clusters were at their 5'-end, via the PCR-reaction, linked to nucleotide sequences (included in SEQ ID NO: 11,

15, 17, 19, 21 and 23) encoding the amino acid sequence SEQ

ID NO: 37 which constitute a cleavage site for the bovine restriction protease FX_a (Nagai and Thogersen, Methods in Enzymology, 152: 461-481, 1987). The amplified DNA fragments were either subcloned into the E. coli expression vector pT₇H₆ (Christensen et al., FEBS Letters. 295: 181-184, 1991) or the expression plasmid pLcIIMLCHg, which is modified from pLcIIMLC (Nagai et al., Nature, 332: 284-286, 1988) by the insertion of an oligonucleotide encoding six histidinyl residues C-terminal of the myosin light chain fragment. The construction of the resulting plasmids pT₇H₆FX-#l to #3 and pLcIIMLCH₆FX-#4 to #9 is outlined in fig. 6-8 and in figure 9 is shown the amino acid sequence of the expressed protein (in SEQ ID NO: 52 is shown the amino acid sequence encoded by the full length reading frame).

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<img file="CA2155335C_D0013.tif" />

The domains and domain-clusters subcloned in the pT₇H₆FX series were grown and expressed in E. coli BL21 cells in a medium scale (2 litre) as described by Studier, and Moffat, J. Mol. Biol., 189: 113-130, 1986. Exponentially growing cultures at 37°C were at OD₆₀₀ 0.8 infected with bacteriophage XCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base).

The domain-clusters subcloned in the pLcIIMLCH₆ series were grown and expressed in E. coli QY13 cells as described in Nagai and Thogersen. Methods in Enzymology, 152: 461-481, 1987. Exponentially growing cultures (4 litre) at 30°C were at ODgQQ 1.0 transferred to 42°C for 15 min. This heat shock induces synthesis of the fusion proteins. The cultures are further incubated at 37°C for three to four hours before cells are harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base).

Crude protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 0.1 M dithioerythrdjfol.^Following gel filtration on Sephadex G-25 (Pharmacia, Sweden) into 8 M Urea, 1 M NaCl, 50 mM Tris-HCl pH 8, 10 mM 2-mercaptoethanol and 2 mM methionine the crude protein preparations were applied to a Ni²⁺ activated NTA-agarose columns for purification (Hochuli et al., 1988) of the fusion proteins and subsequently to undergo the cyclic folding procedure.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

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Preparation and charging” of the Ni²⁺NTA-agarose column is described under Example 1.

Upon application of the crude protein extracts on the Ni²⁺NTA-agarose column, the fusion proteins were purified from the majority of coli and λ phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl, 10 mM 2-mercaptoethanol, and 2 mM methionine until the optical density (OD) at 280 nm of the eluate was stable.

Each of the fusion proteins were refolded on the Ni²⁺NTAagarose column using a gradient manager profile as described in table 4 and 0.5 M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl₂, 0.33 mM methionine, and 2.0 mM/0.2 mM reduced/oxidized gluthatione as buffer A and 4 M urea, 0.5 M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl₂, 2 mM methionine, and 3 mM reduced gluthatione as buffer B. The reduced/oxidized gluthatione solution was freshly prepared as a 100 times stock solution by addition of 9.9 M H₂O₂ to a stirred solution of 0.2 M reduced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure the fusion proteins representing domains and domain-clusters derived from the a₂-MR protein were eluted from the Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, 5 mM EDTA pH 8. Fusion proteins that were aggregated and pre25 cipitated on the Ni²⁺NTA-agarose column were eluted in buffer B.

Approximately 75% of the fusion protein material expressed from the plasmids pT₇H₆FX-#l and #2, representing the Nterminal two and four cysteine-rich domains of the œ₂-MR protein were eluted from the Ni²⁺NTA-agarose column by the non denaturing buffer. The majority of this fusion protein material appeared as monomeric as judged by non reducing SDSPAGE analysis. The yield of monomeric fusion protein #1 and #2 were estimated to approximately 50 mg.

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<img file="CA2155335C_D0014.tif" />

3 û ^J

Approximately 50% of the fusion protein material expressed from all other expression plasmids representing domain-clusters derived from the o?₂-MR protein were eluted from the Ni²⁺NTA-agarose column by the non denaturing buffer. Between 30% (fusion proteins #5 and #V) and 65% (fusion protein #4) of these fusion proteins appeared as monomeric as judged by non reducing SDS-PAGE analysis (see Fig. 17, lanes 9 and 10).

Each fusion protein eluted by the non denaturing buffer was cleaved with the restriction protease night at room temperature in an estimated weight ratio of 100 to one.

elution FX_a overto weight

Upon gelfiltration on Sephadex G-25 into 100 mM NaCl, 25 mM Tris-HCl pH 8, the protein solution was passed through a Ni²⁺NTA-agarose column thereby removing uncleaved fusion protein and the liberated N-terminal fusion tail originating from the cleaved fusion proteins. FX_a was removed from the solution by passing the recombinant protein solutions through a small column of SBTI-agarose (Soy Bean Trypsin Inhibitor immobilized on Sepharose CL-6B (Pharmacia, Sweden)).

SDS-PAGE analysis of the refolded, soluble fusion protein product #4 is presented in fig. 17, lanes 9 and 10, showing reduced and unreduced samples, respectively. The mobility increase observed for the unreduced sample reflects the compactness of the polypeptide due to the presence of 33 disulphide bridges.

Each of the recombinant proteins were found to bind Ca²⁺ in a structure dependent manner.

It was found by Dr. Soren Moestrup that a monoclonal antibody, A2MRa-5 derived from the natural human a₂-MR, bound the recombinant proteins expressed by the constructs #4, #6, and #7 whereas a monospecific antibody, A2MRa-3 derived also from natural a₂-MR, was found to bind the recombinant protein

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PCT/DK94/00054 expressed by construct #8. The binding specificity of both antibodies is structure dependent (i.e. the antibodies do neither react with reduced a₂-MR nor with reduced recombinant protein)

EXAMPLE 5

Production and folding of bovine coagulation Factor X_a (FX_a)

This example describes the production in E. coli of one fragment derived from bovine FX_a as a FX_a cleavable fusion protein and the purification, in vitro folding, and the processing of the recombinant protein.

The cDNA encoding bovine FX was cloned by specific amplification in a Polymerase Chain Reaction (PCR) of the nucleotide sequences encoding bovine FX from amino acid residue Ser₈₂ to Trp₄₈₄ (SEQ ID NO: 2, residues 82-484) (FXAy, amino acid numbering relates to the full coding reading frame) using ls^fcstrand oligo-dT primed cDNA synthesized from total bovine liver RNA as template. Primers used in the PCR were SEQ ID NO: 25 and SEQ ID NO: 26. RNA extraction and cDNA synthesis were performed using standard procedures.

The amplified reading frame encoding FXAy was at the 5¹-end, via the PCR-reaction, linked to nucleotide sequences encoding the amino acid sequence SEQ ID NO: 37 which constitute a cleavage site for the bovine restriction protease FX_a (Nagai, and Thegersen. Methods in Enzymology, 152: 461-481, 1987).

The amplified DNA fragments was cloned into the E. coli expression vector pLcIIMLCHg, which is modified from pLcIIMLC (Nagai et al., Nature, 332: 284-286, 1988) by the insertion of an oligonucleotide encoding six histidinyl residues C-terminal of the myosin light chain fragment. The construction of the resulting plasmid pLcIIMLCHgFX-FXAy is outlined in fig.

and in figure 11 is shown the amino acid sequence of the

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PCT/DK94/00054 expressed protein (in SEQ ID NO: 53 is shown the amino acid sequence encoded by the full length reading frame).

The pLcIIMLCHg-FXAy plasmid was grown and expressed in E. coli QY13 cells as described in Nagai and Th0gersen (Methods in Enzymology, 152: 461-481, 1987). Exponentially growing cultures at 30°C were at OD₆₀₀ 1.0 incubated at 42°C for 15 min. This heat shock induces synthesis of the fusion proteins. The cultures are further incubated at 37°C for three to four hours before cells are harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base).

Crude protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 0.1 M dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 1 M NaCI, 50 mM Tris-HCl pH 8, 10 mM 2-mercaptoethanol the crude protein preparation was applied to a Ni²⁺ activated NTA-agarose matrix for purification (Hochuli et al., 1988.) of the FXAy fusion protein and subsequently to undergo the cyclic folding procedure.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

Preparation and charging of the Ni²⁺NTA-agarose column is described under Example 1.

Upon application of the crude protein extracts on the Ni²⁺NTA-agarose column, the fusion proteins were purified from the majority of coli and λ phage proteins by washing with one column volume of the loading buffer followed by 6 Μ guanidinium chloride, 50 mM Tris-HCl, and 10 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.

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The fusion protein was refolded on the Ni²⁺NTA-agarose column using a gradient manager profile as described in table 5 and 0.5 M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl₂, and 2.0 mM/0.2 mM reduced/oxidized gluthatione as buffer A and 8 M urea, 0.5

M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl₂, and 3 mM reduced gluthatione as buffer B. The reduced/oxidized gluthatione . solution was freshly prepared as a 100 times stock solution by addition of 9.9 M H₂O₂ to a stirred solution of 0.2 M reduced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure the FXAy fusion protein was eluted from the Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, 5 mM EDTA pH 8. Fusion protein that was aggregated and precipitated on the Ni²⁺NTA-agarose column was eluted in buffer B.

Approximately 33% of the FXAy fusion protein material was eluted from the Ni²⁺NTA-agarose column by the non denaturing buffer. The amount of FXAy fusion protein was estimated to 15 mg. Only about one third of this fusion protein material appeared as monomeric as judged by non reducing SDS-PAGE analysis corresponding to an overall efficiency of the folding procedure of approximately 10%.

FXAy fusion protein in non denaturing buffer was activated by passing the recombinant protein solution through a small column of trypsin-agarose (trypsin immobilized on Sepharose

CL-6B (Pharmacia, Sweden)).

The activated recombinant FXAy fusion protein was assayed for proteolytic activity and substrate specificity profile using standard procedures with chromogenic substrates. The activity and substrate specificity profile was indistinguishable from that obtained for natural bovine FX_a

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EXAMPLE 6

Production and folding of kringle domains 1 and 4 from human plasminogen

This example describes the production in E. coli of the lysine binding kringle domains 1 and 4 from human plasminogen (Kl and K4, respectively) as FX_a cleavable fusion proteins and the purification and in vitro folding of the Kl- and K4-fusion proteins.

A plasmid clone containing the full length cDNA encoding human plasminogen cloned into the general cloning vector pUC18 (generously provided by Dr. Earl Davie, Seattle, USA) were used as template in a Polymerase Chain Reaction (PCR) designed to produce cDNA fragments corresponding to Kl (corresponding to amino acid residue Ser_ei to Glu₁₆₂ in so-called Glu-plasminogen) and K4 (corresponding to amino acid residue Val₃₅₄ to Ala₄₃₉ in so-called Glu-plasminogen) . The primers SEQ ID NO: 27 and SEQ ID NO: 28 were used in the PCR producing Kl and the primers SEQ ID NO: 29 and SEQ ID NO: 30 were used in the PCR producing K4.

The amplified reading frames encoding Kl and K4 were at their 5'-ends, via the PCR-reaction, linked to nucleotide sequences, included in SEQ ID NO: 27 and SEQ ID NO: 29, encoding the amino acid sequence SEQ ID NO: 37 which constitute a cleavage site for the bovine restriction protease FX_a (Nagai and Thogersen. Methods in Enzymology, 152:

461-481, 1987). The amplified Kl DNA fragment was cloned into the E. coli expression vector pLcIIMLCHg, which is modified from pLcIIMLC (Nagai et al., Nature, 332: 284-286, 1988) by the insertion of an oligonucleotide encoding six histidinyl residues C-terminal of the myosin light chain fragment. The construction of the resulting plasmid pLcIIMLCHgFX-Kl is outlined in fig. 12. The amplified K4 DNA fragment was cloned into the E. coli expression vector pLcIIHg, which is modified from pLcII (Nagai and Thogersen. Methods in Enzymology, 152;

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<img file="CA2155335C_D0015.tif" />

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461-481, 1987) by the insertion of an oligonucleotide encoding six histidinyl residues C-terminal of the ell fragment. The construction of the resulting plasmid pLcIIH₆FX-K4 is outlined in fig. 13 and in fig. 14 is shown the amino acid sequence of human Glu-plasminogen (SEQ ID NO: 54).

Both the pLcIIMLCHg-Kl plasmid and the pLcIIH₆FX-K4 plasmid were grown and expressed in E. coli QY13 cells as described in Nagai and Thogersen. Methods in Enzymology, 152: 461-481, 1987. Exponentially growing cultures at 30°C were at OD₆₀₀

1.0 transferred to 42°C for 15 min. This heat shock induces synthesis of the fusion proteins. The cultures are further incubated at 37°C for three to four hours before cells are harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base).

Crude protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 0.1 M dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, Sweden) into 8 M Urea, 1 M NaCl, 50 mM Tris-HCl pH 8, 10 mM 2-mercaptoethanol, and 2 mM methionine the crude protein preparation was applied to a Ni²⁺ activated NTA-agarose matrix for purification (Hochuli et al., 1988.) of the Kland K4-fusion proteins and subsequently to undergo the cyclic folding procedure.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

Preparation and charging of the Ni²⁺NTA-agarose column is described under Example 1.

Upon application of the crude protein extracts on the Ni²⁺NTA-agarose column, the fusion proteins were purified from the majority of coli and λ phage proteins by washing

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<img file="CA2155335C_D0016.tif" />

PCT/DK94/00054 with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl,10 mM 2-mercaptoethanol, and 2 mM methionine until the optical density (OD) at 280 nm of the column eluate was stable.

The fusion protein was refolded on the Ni²⁺NTA-agarose column using a gradient manager profile as described in table 4 with 0.5 M NaCl, 50 mM Tris-HCl pH 8, 10 mM 6 aminohexanoic acid (e-aminocapronic acid, e-ACA), 0.33 mM methionine, and 2.0 mM/0.2 mM reduced/oxidized gluthatione as buffer A and 4 M Urea, 0.5 M NaCl, 50 mM Tris-HCl pH 8, 10 mM e-ACA, 2 mM methionine, and 3 mM reduced gluthatione as buffer B. The reduced/oxidized gluthatione solution was freshly prepared as a 100 times stock solution by addition of 9.9 M H₂0₂ to a stirred solution of 0.2 M reduced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure each of the

Kl- and K4 fusion proteins were eluted from the

Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, mM Tris-HCl, 5 mM EDTA pH 8. Fusion proteins that were aggregated and precipitated on the Ni²⁺NTA-agarose column was eluted in buffer B.

Virtually all of the Kl- and K4-fusion protein material were eluted from the Ni²⁺NTA-agarose columns by the non denaturing buffer. The estimated yield of Kl-fusion protein and K4-fusion protein were approximately 60 mg. Virtually all of the Kl-fusion protein as well as the K4-fusion protein appeared as monomeric as judged by non reducing SDS-PAGE analysis corresponding to an efficiency of the folding procedure above 90%.

SDS-PAGE analysis of the production of recombinant plasminogen kringles 1 and 4 is presented in fig. 17.

The Kl-fusion protein and the K4-fusion protein were further purified by affinity chromatography on lysine-Sepharose CL-6B

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PCT/DK94/0O054 (Pharmacia, Sweden). The fusion proteins were eluted from the affinity columns by a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8, 10 mM e-ACA.

Binding to lysine-Sepharose is normally accepted as indica5 tion of correct folding of lysine binding kringle domains.

The three dimensional structure of recombinant K1 and K4 protein domains, produced by this cyclic folding procedure and which have been fully processed by liberation from the Nterminal fusion tail and subsequently purified by ion exchange chromatography, have been confirmed by X-ray diffraction (performed by Dr. Robert Huber) and two dimensional NMR analysis (performed by stud, scient. Peter Reinholdt and Dr. Flemming Poulsen).

The general yield of fully processed recombinant K1 and K4 protein domains by this procedure is 5 mg/litre culture.

EXAMPLE 7

Production in E. coli and refolding of recombinant fragments derived from human &amp;₂-Macroglubolin and chicken Ovostatin

This example describes the production in E. coli of the receptor-binding domain of human σ₂-Macroglobulin (œ₂-MRBDv) as a FX_a cleavable fusion protein, and the purification of the recombinant a₂^_MRBDv after FX_a cleavage.

The 4 62 bp DNA fragment encoding the a₂-Macroglobulin reading frame from amino acid residue Val₁₂₉₉ to Ala₁₄₅₁ (œ₂-MRDv) was amplified in a Polymerase Chain Reaction (PCR), essentially following the protocol of Saiki et al., (1988). pA2M (generously provided by Dr. T. Kristensen) containing the full length cDNA of human a₂-Macroglobulin was used as template, and the oligonucleotides SEQ ID NO: 31 and SEQ ID NO: 32 as primers. The amplified coding reading frame was at the

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2155335 es

5'-end, via the PCR-reaction, linked to a nucleotide sequence, included in SEQ ID NO: 7, encoding the amino acid sequence SEQ ID NO: 37 which constitute a cleavage site for the bovine restriction protease FX_a (Nagai ana Thogersen,

1987). The amplified DNA fragment was subcloned into the E. cold, expression vector pT₇H₆ (Christensen et al., 1991) . The construction of the resulting plasmid pT₇H₆FX-oi2MRDv (expressing human a₂-MRDv) is outlined in fig. 18 and the amino acid sequence of the expressed protein is shown in fig. 19 (SEQ ID NO: 55).

Recombinant human œ₂MRDv was produced by growing and expressing the plasmid pT₇H₆FX-a₂MRDv in E. cold BL21 cells in a medium scale (2x1 litre) as described by Studier and Moffat,

J. Mol. Biol., 189: 113-130, 1986. Exponentially growing cultures at 37°C were at OD₆₀₀ 0.8 infected with bacteriophage XCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and Bonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 1 M NaCl, 50 mM Tris-HCl pH 8,and 10 mM 2-mercaptoethanol the crude protein preparation was applied to a Ni²⁺ activated NTA-agarose column (Ni²⁺NTA- agarose) for purification (Hochuli et al., 1988) of the fusion protein, MGSHHHHHHGSIEGR-tt₂MRDv (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) and subsequently to undergo the cyclic folding procedure.

Preparation and charging of the Ni²⁺NTA-agarose column is described under Example 1.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

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Upon application of the crude protein extract on the Ni²⁺NTAagarose column, the fusion protein, MGSHHHHHHGSIEGR-œ₂MRDv (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) was purified from the majority of coli and λ phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl, and 10 mM 2-mercaptoethanol, until the optical density (OD) at 280 nm of the eluate was stable.

The fusion protein was refolded on the Ni²⁺NTA-agarose column 10 using a gradient manager profile as described in table 4 and

0.5 M NaCl, 50 mM Tris-HCl pH 8, and 2.0 mM/0.2 mM reduced/oxidized gluthatione as buffer A and 8 M urea, 0.5 M NaCl, 50 mM Tris-HCl pH 8, and 5 mM reduced gluthatione as buffer B. The reduced/oxidized gluthatione solution was freshly prepared as a 200 times stock solution by addition of 9.9 M H₂O₂ to a stirred solution of 0.2 M reduced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure the ûî₂MRDv fusion protein was eluted from the Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, 20 mM EDTA pH 8. Fusion protein that were aggregated and precipitated on the Ni²⁺NTA-agarose column was eluted in buffer B.

Approximately 50% of the fusion protein material was eluted in the aqueous elution buffer. Half of this fusion protein material appeared monomeric and folded as judged by non-reducing SDS-PAGE analysis.

Recombinant a₂MRDv protein was liberated from the N-terminal fusion tail by cleavage with the restriction protease FX_a at room temperature in a weight to weight ratio of approximately

50 to one for four hours. After cleavage the a₂MRDv protein was isolated from uncleaved fusion protein, the liberated fusion tail, and FX_a, by gelfiltration on Sephadex G-25 into 10 mM NaCl, 50 mM Tris-HCl pH 8, followed by ion exchange chromatography on Q-Sepharose: ce₂MRDv was eluted in a linear

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PCT/DK94/00054 gradient (over 10 column volumes) from 10 mM NaCl, 10 mM Tris-HCl pH 8 to 500 mM NaCl·, 10 mM Tris-HCl pH 8. The a₂MRDv protein eluted at 150 mM NaCl.

The recombinant œ₂MRDv domain binds to the œ₂M-receptor with a similar affinity for the receptor as exhibited by the complete a₂-Macroglobulin molecule (referring to the estimated K_D in one ligand-one receptor binding (Moestrup and Gliemann 1991)). Binding analysis was performed by Dr. Soren

K. Moestrup and stud, scient. Rare Lehmann).

EXAMPLE 8

Production in E. coli and refolding- of recombinant fragments derived from the trout virus VHS envelope glycoprotein G

Expression and in vitro refolding of recombinant fragments derived from the envelope glycoprotein G from the trout virus VHS in E. coli as FX_a cleavable fusion proteins is performed using general strategies and methods analogous to those outlined in the general description of the cyclic refolding procedure and given in Examples 1 through 6.

EXAMPLE 9

Production in E. coli and refolding of recombinant human Tetranectin and recombinant fragments derived from human Tetranectin

Tetranectin is a tetrameric protein consisting of four identical and non-covalently linked single chain subunits of 181 amino acid residues (17 kDa). Each subunit contains three disulphide bridges and binds Ca²⁺. Tetranectin is found in plasma and associated with extracellular matrix. Tetranectin binds specifically to plasminogen kringle 4. This binding can be specifically be titrated by lysine or ω-amino acids.

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The cDNA encoding the reading frame corresponding to the mature tetranectin single chain subunit was cloned by specific amplification in a Polymerase Chain Reaction (PCR) (Saiki et al., 1988) of the nucleotide sequences from amino acid residue Glu_x to Val₁₈₁ using Is¹¹ strand oligo-dT primed cDNA synthesized from total human placental RNA as template. Primers used in the PCR were SEQ ID NO: 33 and SEQ ID NO: 34. RNA extraction and cDNA synthesis were performed using standard procedures.

The amplified reading frame encoding the monomer subunit of tetranectin was at the 5'-end, via the PCR-reaction, linked to nucleotide sequences encoding the amino acid sequence SEQ ID NO: 37 which constitute a cleavage site for the bovine restriction protease FX_a (Nagai, and Thogersen, 1987). A glycine residue was, due to the specific design of the 5'-PCR primer (SEQ. ID NO. 33), inserted between the C-terminal arginine residue of the FX_a cleavage site (SEQ ID NO. 37) and the tetranectin Glu_x-residue. The amplified DNA fragment was subcloned into the E. coli expression vector pT₇H₆ (Christensen et al., 1991). The construction of the resulting plasmid pT₇H₆FX-TETN (expressing the tetranectin monomer) is outlined in fig. 20 and the amino acid sequence of the expressed protein is shown in fig, 21 (in SEQ ID NO: 56 is shown the amino acid sequence encoded by the full length reading frame) .

To prepare the tetranectin monomer, the plasmid pT₇H₆FX-TETN was grown in medium scale (4x1 litre; 2xTY medium, 5 mM MgS0₄ and 100 μg ampicillin) in E. coli BL21 cells, as described by Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986. Exponentially growing cultures at 37°C were at OD₆₀₀0.8 infected with bacteriophage XCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours and the cells harvested by centrifugation. Cells were resuspended in 150 ml of 0.5 M NaCl, 10 mM Tris-HCl pH 8, and 1 mM EDTA pH 8. Phenol (100 ml adjusted to pH 8) was added and the mixture sonicated to extract the total protein.

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Protein was precipitated from the phenol phase by 2.5 volumes of ethanol and centrifugation.

The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 0.1 M dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 1 M NaCl, 50 mM Tris-HCl pH 8 and 10 mM 2-mercaptoethanol, the crude protein preparation was applied to a Ni²⁺ activated NTA-agarose column (Ni²⁺NTA-agarose, 75 ml pre-washed with 8 M urea, 1 M NaCl, mM Tris-HCl pH 8, and 10 mM 2-mercaptoethanol) for purification (Hochuli et al., 1988) of the fusion protein, MGSHHHHHHGSIEGR-TETN (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) .

Preparation and charging” of the Ni²⁺NTA-agarose column is described under example 1.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

The column was washed with 200 ml of 8 M urea, 1 M NaCl, 50 mM Tris-HCl pH 8, and 10 mM 2-mercaptoethanol (Buffer I) and 100 ml 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 10 mM 2-mercaptoethanol (Buffer II). The MGSHHHHHHGSIEGR-TETN fusion protein was eluted with Buffer II containing 10 mM EDTA pH 8 and the elute was gel filtered on Sephadex G25 using Buffer I as eluant.

The protein eluted was then refolded. The fusion protein MGSHHHHHHGSIEGR-TETN (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) was mixed with 100 ml Ni²⁺NTA-agarose. The resin containing bound protein was packed into a 5 cm diameter column and washed with Buffer I supplemented with CaCl2 to 2 mM. The fusion protein was refolded on the Ni²⁺NTA-agarose column at 11-12°C using a gradient manager profile as described in table 4 and 0.5 M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl2 and 2.0 mM/0.2 mM reduced/oxidized gluthatione as buffer A and 8 wo 94/18227 ' 21 5 5 3 3 5

PCT/DK94/00054

M urea, 1 M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl₂ and 3 mM reduced gluthatione as buffer 3. The reduced/oxidized gluthatione solution was freshly prepared as a 200 times stock solution by addition of 9.9 M H₂O₂ to a stirred solution of

0.2 M reuuced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure the tetranectin fusion protein was eluted from the Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, mM EDTA pH 8. The tetranectin fusion protein was cleaved with FX_a at 4°C overnight in a molar ratio of 1:300. After FX_a cleavage the protein sample was concentrated 10 fold by , ultrafiltration on a YM10 membrane (Amicon) . Recombinant tetranectin was, after ten times dilution of the protein sample with 2 mM CaCl₂, isolated by ion-exchange chromato15 graphy on Q-Sepharose (Pharmacia, Sweden) in a liner gradient over 10 column volumes from 10 mM Tris-HCl pH 8, 2 mM CaCl₂to 10 mM Tris-HCl pH 8, 2 mM CaCl₂, and 0.5 M NaCl.

Recombinant tetranectin produced by this procedure was analyzed by Dr. Inge Clemmensen Rigshospitalet, Copenhagen. Dr.

Clemmensen found that the recombinant tetranectin with respect to binding to plasminogen kringle 4 and expression of antigenic sites behaved identically to naturally isolated human tetranectin.

)

Preliminary experiments comparing the efficiency of refold25 ing, using the cyclic refolding procedure, of recombinant Tetranectin fusion protein bound to the Ni²⁺NTA-agarose column versus recombinant Tetranectin contained in a dialysis bag indicate a significantly improved yield of soluble monomer from the solution refolding strategy. However, if either product of the cycling procedures is subjected to disulphide re-shuffling in solution in the presence of 5 mM CaCl₂ virtually all of the polypeptide material is converted to the correctly folded Tetranectin tetramer.

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Denatured and reduced recombinant authentic Tetranectin contained in a dialysis bag, was refolded over 15 cyclic exposures to buffer B (6 M Urea, 100 mM Nacl, 50 mM Tris-HCl pH=8, 2 mM/0.2 mM reduced/oxidized glutathione, 2 mM CaCl₂and 0.5 mM methionine) and buffer A (100 mM NaCl·, 50 mM Tris-HCl pH 8, 2 mM/0.2 mM reduced/oxidized glutathione, 2 mM CaCl₂f and 0,5 mM methionine).

EXAMPLE 10

Production and folding of a diabody expressed intracellularly in E. coli: Mab 32 diabody directed against tumour necrosis factor.

Diabodies (described in Holliger et al., 1993) are artificial bivalent and bispecific antibody fragments.

This example describes the production in E. coli of a diabody directed against tumour necrosis factor alpha (TNF-α), derived from the mouse monoclonal antibody Mab 32 (Rathjen et al., 1991, 1992; Australian Patent Appl. 7,576;

EP-A-486,526).

A phagemid clone, pCANTAB5-myc-Mab32-5, containing Mab32 encoded in the diabody format (PCT/GB93/02492) was generously provided by Dr. G. Winter, Cambridge Antibody Technology (CAT) Ltd., Cambridge, UK. pCANTAB5-myc-Mab32-5 DNA was used as template in a Polymerase Chain Reaction (PCR) (Saiki et al., 1988), using the primers SEQ ID NO: 35 and SEQ ID NO:

36, designed to produce a cDNA fragment corresponding to the complete artificial diabody. The amplified coding reading frame was at the 5'-end, via the PCR-reaction, linked to a nucleotide sequence, included in SEQ ID NO: 35, encoding the amino acid sequence SEQ ID NO: 37 which constitute a cleavage site for the bovine restriction protease FX_a (Nagai and Thogersen, 1987) . The amplified DNA fragment was subcloned into the E. coli expression vector pT₇H₆ (Christensen et al., 1991). The construction of the resulting plasmid pT₇H₆FX-DB32

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<img file="CA2155335C_D0017.tif" />

(expressing the Mab32 diabody) is outlined in fig. 22 and the amino acid sequence of the expressed protein is shown in fig. 23 (in SEQ ID NO: 57 is shown the amino acid sequence encoded by the full length reading frame).

To prepare the diabody fragment, the plasmid pT₇H₆FX-DB32 was grown in medium scale (4x1 litre; 2xTY medium, 5 mM MgSO₄and 100 /xg ampicillin) in E. coli BL21 cells, as described by Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986. Exponentially growing cultures at 37°C were at OD₆₀₀ 0.8 infected with bacteriophage ÂCE6 at a multiplicity of approximately 5. Forty minutes after infection, rifampicin was added (0.2 g in 2 ml methanol per litre media). Cultures were grown at 37°C for another three hours and the cells harvested by centrifugation. Cells were resuspended in 150 ml of 0.5 M

NaCI, 10 mM Tris-HCl pH 8, and 1 mM EDTA pH 8. Phenol (100 ml adjusted to pH 8) was added and the mixture sonicated to extract the total protein. Protein was precipitated from the phenol phase by 2.5 volumes of ethanol and centrifugation.

The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 0.1 M dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 1 M NaCI, 50 mM Tris-HCl pH 8 and 10 mM 2-mercaptoethanol, the crude protein preparation was applied to a Ni²⁺ activated NTA-agarose column (Ni²⁺NTA-agarose, 75 ml pre-washed with 8 M urea, 1 M NaCI, mM Tris-HCl pH 8, and 10 mM 2-mercaptoethanol) for purification (Hochuli et al., 1988) of the fusion protein,

MGSHHHHHHGSIEGR-DB32 (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) .

0 Preparation and charging of the Ni²⁺NTA-agarose column is described under example 1.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

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2^55335 96

The column was washed with 200 ml of 8 M urea, 1 M NaCl, 50 mM Tris-HCl pH 8, and 10 mM 2-mercaptoethanol (Buffer I) and 100 ml 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 10 mM 2-mercaptoethanol (Buffer II). The MGSHHHHHHGSIEGR-DB32 fusion protein was eluted with Buffer II containing 10 mM EDTA pH 8 and the elute was gel filtered on Sephadex G25 using Buffer I as eluant.

The protein eluted was then refolded.The fusion protein MGSHHHHHHGSIEGR-DB32 (wherein MGSHHHHHHGSIEGR is SEQ ID NO:

48) was mixed with 100 ml Ni²⁺NTA-agarose. The resin containing bound protein was packed into a 5 cm diameter column and washed with Buffer X. The fusion protein was refolded on the Ni²⁺NTA-agarose column at 11-12°C using a gradient manager profile as described in table 4 and 0.5 M NaCl, 50 mM

Tris-HCl pH 8, and 2.0 mM/0.2 mM reduced/oxidized gluthatione as buffer A and 8 M urea, 1 M NaCl, 50 mM Tris-HCl pH 8, and 3 mM reduced gluthatione as buffer B. The reduced/oxidized gluthatione solution was freshly prepared as a 200 times stock solution by addition of 9.9 M H₂0₂ to ^a stirred so20 lution of 0.2 M reduced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure the DB32 fusion protein was eluted from the Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, 25 mM

EDTA pH 8 and adjusted to 5 raM GSH, 0.5 mM GSSG and incubated for 12 to 15 hours at 20°C. The fusion protein was then concentrated 50 fold by ultrafiltration using YM10 membranes and clarified by centrifugation.

The DB32 fusion protein dimer was purified by gel filtration using a Superose 12 column (Pharmacia, Sweden) with PBS as eluant.

The overall yield of correctly folded DB32 fusion protein from this procedure was 4 mg per litre.

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Tin analysis by non-reducing SDS-PAGE from different stages of the purification is shown in fig. 26.

The MGSHHHHHHGSIEGR (SEQ ID NO: 48) N-terminal fusion peptide was cleaved off the DB32 protein by cleavage with tne restriction protease FX_a (molar ratio 1:5 FX_a:DB32 fusion protein) at 37°C for 20 hours. This is shown as the appearance of a lower molecular weight band just below the uncleaved fusion protein in fig. 26.

The refolded DB32 protein was analyzed by Cambridge Antibody Technology Ltd. (CAT). DB32 was found to bind specifically to TNF-o; and to compete with the Mab32 whole antibody for binding to TNF-α. Furthermore both DB32 and Mab32 were competed in binding to TNF-o; by sheep anti-301 antiserum, which has been raised by immunizing sheep with a peptide encoding the first 18 amino acids of human TNF-œ and comprise at least part of the epitope recognised by the murine Mab32.

EXAMPLE 11

Production and refolding of human psoriasin in E. coli.

Psoriasin is a single domain Ca²⁺- binding protein of 100 amino acid residues (11.5 kDa). Psoriasin contains a single disulphide bridge. The protein which is believed to be a member of the S100 Protein family is highly up-regulated in psoriatic skin and in primary human kératinocytes undergoing abnormal differentiation.

The plasmid pT₇H_gFX-PS.4 (kindly provided by Dr. P. Madsen, Insitute of Medical Biochemistry, University of Aarhus, Denmark) has previously been described by Hoffmann et al., (1994). The nucleotide sequence encoding the psoriasin protein from Ser₂ to Gln₁₀₁ is in the 5'-end linked to the nucleotide sequence encoding the amino acid sequence MGSHHHHHHGSIEGR (SEQ ID NO: 48). A map of pT₇H_gFX-PS.4 is given in fig. 24 and the amino acid sequence of human psoria94/18227

PCT/DK94/00054 f- ^ç 9 ' A ?} ό û J tj sin is listed in fig. 25 acid sequence encoded by (in SEQ ID NO: 58 is shown the amino the full length reading frame).

Recombinant human psoriasin was grown and expressed from the plasmid pT₇H₆FX~PS.4 in E. coli BL21 cells and total cellular protein extracted as described (Hoffmann et al., 1994). Ethanol precipitated total protein was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 0,5 M NaCl, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol the crude protein preparation was applied to a Ni²⁺ activated NTA-agarose column (Ni²⁺NTA-agarose) for purification (Hochuli et al.,

1988) of the fusion protein, MGSHHHHHHGSIEGR-psoriasin (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) and subsequently to undergo the cyclic folding procedure.

Preparation and charging of the Ni²⁺NTA-agarose column is described under Example 1.

All buffers prepared for liquid chromatography were degassed under vacuum prior to addition of reductant and/or use.

Upon application of the crude protein extract on the Ni²⁺NTAagarose column, the fusion protein, MGSHHHHHHGSIEGR-psoriasin (wherein MGSHHHHHHGSIEGR is SEQ ID NO: 48) was purified from the majority of coli and λ phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl, and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.

The fusion protein was refolded on the Ni²⁺NTA-agarose column using a gradient manager profile as described in table 4 and 0.5 M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl₂ and 1.0 mM/0.1 mM reduced/oxidized gluthatione as buffer A and 8 M urea, 0.5 M NaCl, 50 mM Tris-HCl pH 8, 2 mM CaCl₂ and 5 mM reduced gluthatione as buffer B. The reduced/oxidized gluthatione so94/18227

PCT/DK94/00054 lution was freshly prepared as a 200 times stock solution by addition of 9.9 M H₂0₂ to a stirred solution of 0.2 M reduced gluthatione before addition to buffer A.

After completion of the cyclic folding procedure the psoriasin fusion protein was eluted from the Ni²⁺NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl, mM EDTA pH 8. Fusion protein that were aggregated and precipitated on the Ni²⁺NTA-agarose column was eluted in buffer B.

Approximately 95% of the fusion protein material was eluted by the non denaturing elution buffer. As judged by non-reducing SDS-PAGE analysis 75% of the soluble fusion protein material appeared to be monomeric yielding an overall efficiency of the folding procedure of approximately 70%. The efficiency of the previously described refolding procedure for production of recombinant human psoriasin (Hoffman et al., 1994) was estimated to be less than 25%.

The psoriasin fusion protein was cleaved with FX_a in a molar ratio of 100:1 for 48 hrs at room temperature. After gelfiltration into a buffer containing 20 mM Na-acetate pH 5 and 20 mM NaCl on Sephadex G-25 the protein sample was applied onto a S-Sepharose ion exchange column (Pharmacia). Monomeric recombinant psoriasin was eluted over 5 column volumes with a linear gradient from 20 mM Na-acetate pH 5, 20 mM NaCl to 0.5 M NaCl. Monomeric psoriasin eluted at 150 mM NaCl. Dimeric and higher order multimers of psoriasin together with uncleaved fusion protein eluted lated in the gradient. Fractions containing the cleaved purified recombinant protein was gelfiltrated on Sephadex G25 into a buffer containing 150 mM NaCl, 10 mM Tris-HCl pH 7.4 and stored at 4°C.

EXAMPLE 12

Evaluation procedure for suitability testing of thiol compounds for use as reducing agents in cyclic refolding and

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100 determination of optimal levels of dénaturants and disulphide reshuffling agents for optimization of cyclic refolding procedures.

In order to improve the yield of correctly folded protein obtainable from cyclic refolding the number of productive cycles should be maximized (see SUMMARY OF THE INVENTION). Productive cycles are characterized by steps of dénaturation where misfolded protein, en route to dead-end aggregate conformational states, is salvaged into unfolded conformational states while most of the already correctly folded protein remains in conformational states able to snap back into the refolded state during the refolding step of the cycle.

A number of disulphide bridge containing proteins, like β₂microglobulin, are known to refold with high efficiency (>95%) when subjected to high levels of denaturing agents as long as their disulphide bridges remain intact.

This example describes how to evaluate suitability of a thiol compound for use in cyclic refolding on the basis of its ability to discriminate correct from incorrect disulphide bridges and how to optimize levels of denaturing agent and/or reducing agent to be used in the dénaturation steps in order to maximize the number of productive cycles. As model system we chose a mixture of mono-, di- and multimeric forms of purified recombinant human £₂^mï^crogl°kulin. Our specific aim was to analyze the stability of different topological forms of human jS₂-microglobulin against reduction by five different reducing agents at various concentrations of denaturing agent.

Human /3₂-microglobulin (produced as described in Example 13) in 6 M guanidinium chloride, 50 mM Tris-HCl and 10 mM 2mercaptoethanol pH 8 was gelfiltrated into non-denaturing buffer (50 mM Tris-HCl, 0.5 M NaCl pH 8). Only a fraction of the protein in the sample was soluble in the non-denaturing buffer. After 48 hours exposure to air, the protein solution

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101 appeared unclear. Non-reducing SDS-PAGE analysis showed that most of the protein had been oxidized into multimeric forms and only a small fraction was oxidized and monomeric (Fig.

27, lane 1).

The protein solution was aliquoted into a number of tubes and varying amounts of urea added while keeping the concentration of protein and salt at a constant level.

Reducing agent, either gluthatione, cysteine ethyl ester, Nacetyl-L-cysteine, mercaptosuccinic acid or 2-mercaptoethanol was added to the ensemble of protein samples with varying urea concentrations. Each reducing agents was added to a final concentration of 4 mM. The protein samples were incubated at room temperature for 10 min and then free thiol groups were blocked by addition of iodoacetic acid to a final concentration of 12 mM. Finally, the protein samples were analyzed by non-reducing SDS-PAGE (fig. 27 - 32). The compositions of the test-samples used in the non-reducing SDS-PAGE as well as the results are given below in the following tables; in the rows indicating the ability of the chosen reducing agent to reduce disulphide bridges the marking +++ indicates good ability, ”++ indicates intermediate ability, + indicates weak ability, whereas no marking indicates that no measurable effect could be observed.

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102

Composition of samples used in SDS-PAGE of fig. 27

<td> Test no.</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 11</td>

<td> μ[ protein solution</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td>

<td> μϊ Buffer A</td><td> 160</td><td> 160</td><td> 140</td><td> 120</td><td> 100</td><td> 80</td><td> 70</td><td> 60</td><td> 50</td><td> 40</td><td> 20</td>

<td> 5 μϊ Buffer B</td><td> 0</td><td> 0</td><td> 20</td><td> 40</td><td> 60</td><td> 80</td><td> 90</td><td> 100</td><td> 110</td><td> 120</td><td> 140</td>

<td> μ.1 GSH</td><td> 0</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td>

<td> M urea</td><td> 0</td><td> 0</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 4.5</td><td> 5</td><td> 5.5</td><td> 6</td><td> 7</td>

<td> Ability to reduce</td><td></td><td></td><td> +</td><td> +</td><td> + +</td><td> + +</td><td> + + +</td><td> + + +</td><td> + + +</td><td> + + +</td><td> + + +</td>

wrong disulphide 10 bridges

Ability to reduce correct disulphide bridges

Buffer A : 50 mM Tris.HCl pH 8, 0.5 M NaCl

Buffer B: 10 M urea, 50 mM Tris.HCl pH 8, 0.5 M NaCl

GSH: 0.2 M Gluthatione

Protein solution: 2 mg/ml h3₂^m, 50 mM Tris.HCl pH 8, 0.5 M NaCl

Composition of samples used in SDS-PAGE of fig. 28

<td> Test no.</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td>

<td> gl protein solution</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td>

<td> μϊ Buffer A</td><td> 160</td><td> 160</td><td> 140</td><td> 120</td><td> 100</td><td> 80</td><td> 60</td><td> 40</td><td> 20</td>

<td> μΐ Buffer B</td><td> 0</td><td> 0</td><td> 20</td><td> 40</td><td> 60</td><td> 80</td><td> 100</td><td> 120</td><td> 140</td>

<td> μΐ CE</td><td> 0</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td>

<td> M urea</td><td> 0</td><td> 0</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td> Ability to reduce wrong disulphide bridges</td><td></td><td> + +</td><td> + +</td><td> + +</td><td> + + +</td><td> + + +</td><td> + + +</td><td> + + +</td><td> + + +</td>

<td> Ability to reduce correct disulphide bridges</td><td></td><td></td><td></td><td></td><td></td><td></td><td> + +</td><td> + + +</td><td> + + +</td>

Buffer A : 50 mM Tris.HCl pH 8, 0.5 M NaCl

0 Buffer B: 10 M urea, 50 mM Tris.HCl pH 8, 0.5 M NaCl

CE: 0.2 M L-cysteine ethyl ester

Protein solution: 2 mg/ml h0₂m, 50 mM Tris.HCl pH 8, 0.5 M NaCl

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103

Composition of samples used in SDS-PAGE of fig. 29

<td> Test no.</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td>

<td> μΐ protein solution</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td>

<td> μΐ Buffer A</td><td> 160</td><td> 160</td><td> 140</td><td> 120</td><td> 100</td><td> 80</td><td> 60</td><td> +G</td><td> 20</td>

<td> μΐ Buffer B</td><td> 0</td><td> 0</td><td> 20</td><td> 40</td><td> 60</td><td> 80</td><td> 100</td><td> 120</td><td> 140</td>

<td> μΐ ME</td><td> 0</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td>

<td> M urea</td><td> 0</td><td> 0</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td> Ability to reduce wrong disulphide bridges</td><td></td><td> + +</td><td> + +</td><td> + +</td><td> + + +</td><td> + + +</td><td> + + +</td><td> + + +</td><td> + + +</td>

<td> Ability to reduce correct disulphide bridges</td><td></td><td></td><td></td><td></td><td></td><td> +</td><td> + +</td><td> + + +</td><td> + + +</td>

Buffer A : 50 mM Tris.HCl pH 8, 0.5 M NaCl

Buffer B: 10 M urea, 50 mM Tris.HCl pH 8, 0.5 M NaCl

ME: 0.2 M 2-mercaptoethanol

Protein solution: 2 mg/ml h/?₂m, 50 mM Tris.HCl pH 8, 0.5 M NaCl

Composition of samples used in SDS-PAGE of fig. 30

<td> Test no.</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td>

<td> μΐ protein solution</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td>

<td> μΐ Buffer A</td><td> 160</td><td> 160</td><td> 140</td><td> 120</td><td> 100</td><td> 80</td><td> 60</td><td> 40</td><td> 20</td>

<td> μΐ Buffer B</td><td> 0</td><td> 0</td><td> 20</td><td> 40</td><td> 60</td><td> 80</td><td> 100</td><td> 120</td><td> 140</td>

<td> μ] MSA</td><td> 0</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td>

<td> M urea</td><td> 0</td><td> 0</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td> Ability to reduce wrong disulphide bridges</td><td></td><td> + +</td><td> + +</td><td> + +</td><td> + +</td><td> + +</td><td> + + +</td><td> + + +</td><td> + + +</td>

<td> Ability to reduce correct disulphide bridges</td><td></td><td></td><td></td><td></td><td></td><td></td><td> + +</td><td> + + +</td><td> + + +</td>

Buffer A : 50 mM Tris.HCl pH 8, 0.5 M NaCl

Buffer B: 10 M urea, 50 mM Tris.HCl pH 8, 0.5 M NaCl

MSA: 0,2 M Mercaptosuccinic acid

Protein solution: 2 mg/ml h0₂m, 50 mM Tris.HCl pH 8, 0.5 M NaCl

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104

Composition of samples used in SDS-PAGE of fig. 31

<td> Test no.</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td>

<td> μΐ protein solution</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td><td> 36</td>

<td> μΐ Buffer A</td><td> 160</td><td> 160</td><td> 140</td><td> izU</td><td> 100</td><td> 80</td><td> 60</td><td> 40</td><td> 20</td>

<td> μΐ Buffer B</td><td> 0</td><td> 0</td><td> 20</td><td> 40</td><td> 60</td><td> 80</td><td> 100</td><td> 120</td><td> 140</td>

<td> μΐ AC</td><td> 0</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td><td> 4</td>

<td> M urea</td><td> 0</td><td> 0</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td> Ability to reduce wrong</td><td></td><td> +</td><td> + +</td><td> + +</td><td> + + +</td><td> + + +</td><td> + + +</td><td> + + +</td><td> + + +</td>

<td> disulphide bridges</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Ability to reduce correct</td><td></td><td></td><td></td><td></td><td> +</td><td> + +</td><td> + + +</td><td> + + +</td><td> + + +</td>

<td> disulphide bridges</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

Buffer A 50 mM Tris.HCl pH 8. 0.5 M NaCl

Buffer B: 10 M urea, 50 mM Tris.HCl pH 8, 0.5 M NaCl

AC: 0.2 M N-acetyl-L-cysteine

Protein solution: 2 mg/ml h/3₂m, 50 mM Tris.HCl pH 8, 0.5 M NaCl

The different topological forms of /3₂'^m be separated by non-reducing SDS-PAGE gel electrophoresis. The fastest migrating band represents the oxidized monomeric form. This band is immediately followed by the reduced β₂“^m with ^a slightly slower migration rate, whereas the multimeric forms of the protein are migrating much slower in the gel.

In this analysis we are probing for the ability of each of the five reducing agents tested, to reduce the disulphide bridges of multimeric forms of /3₂^_in^^cro9^lot)ul^ⁿ without significantly reducing the correctly formed disulphide bridge of the monomeric oxidized form.

The results from the analyses (fig. 27 - 32) are, in summary, as follows: N-acetyl-L-cysteine and mercaptosuccinic acid are, under the conditions used, essentially unable to dis30 criminate correct and incorrect disulphide bridges.

Glutathione, cysteine ethyl ester and 2-mercaptoethanol are all capable of - within 10 min and within individual characteristic ranges of urea concentrations - significantly reducing disulphide bridges of multimeric forms while most of

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105 the oxidised monomeric 0₂-m remains in the oxidised form. Gluthatione has clearly the capacity of selectively reducing incorrect disulphide bridges at higher concentrations of urea compared to cysteine ethyl ester and 2-mercaptoethanol and therefore glutnatione among the selection of thiols tested would be the reducing agent of choice for cyclic refolding of human β₂-microglobulin. As a consequence of these experiments the concentration of urea in the reducing buffer B for the refolding procedure used in Example 13 was lowered from 8 M (Example 1) to 6 M, which led to an improvement of overall refolding yield of human 0₂^mi^cro9l^okulin from 53% to 87%..

EXAMPLE 13

Refolding of purified human β₂_ microglobulin : Comparative analysis of three refolding procedures

The following set of experiments were undertaken to obtain comparable quantitative data to evaluate the importance of cycling for refolding yield versus simple refolding procedures involving a stepwise or a gradual one-pass transition from strongly denaturing and reducing conditions to non20 denaturing and non-reducing conditions.

Purified refolded recombinant human /?₂^mi^croBl°b^ul3-ⁿ fusion protein, obtained as described in EXAMPLE 1, was reduced and denatured to obtain starting materials devoid of impurities, such as proteolytic breakdown products or minor fractions of fusion protein damaged by irreversible oxidation or other chemical derivatization.

In a first step the optimization procedure described in EXAMPLE 12 was used to modify the conditions for cyclic refolding described in EXAMPLE 1 to increase the number of productive cycles. The optimized refolding protocol was identical to that described in EXAMPLE 1, as were buffers and other experimental parameters, except that the Buffer B in

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0.5 M NaCl, 4 mM glutathione.

Three batches of pure fusion protein were refolded while attached to Ni⁺⁺ -loaded NTA-agarose as described in EXAMPLE

1, using the present Buffer B composition. One batch was submitted to buffer cycling as described in EXAMPLE 1, for batch two and three cycling was replaced by a monotonous linear buffer gradient (100% B to 0% B over 24 hours) and a step gradient (100% B to 0% B in one step, followed by 0% B buffer for 24 hours), respectively. In each refolding experiment all of the polypeptide material was recovered as described in EXAMPLE 1 as a soluble fraction elutable under non-denaturing conditions and a remaining insoluble fraction elutable only under denaturing and reducing conditions. The yields of correctly folded fusion protein were the measured by quantitative densitométrie analysis (Optical scanner HW and GS-370 Densitométrie Analysis SW package from Hoeffer Scientific, CA, USA) of Coomassie stained SDS-PAGE gels on which suitably diluted measured aliquots of soluble and insoluble fractions had been separated under reducing or nonreducing condition, as required to allow separation of correctly disulphide-bridged monomer from soluble polymers in soluble fractions. Where required to obtain reliable densitométrie data both for intense and faint bands in a gel lane several sample dilutions were scanned and analyzed to obtain rescaled data sets.

Experimental details and results

Purified denatured and reduced fusion protein:

A batch of human β₂-microglobulin fusion protein was refolded as described in EXAMPLE 1. 96% of the fusion protein was recovered in the soluble fraction (Fig 32, lanes 2-5). 56% of this soluble fraction was in the monomeric and disulphidebridged form. Hence, the overall refolding efficiency obtained was 53%. Monomeric fusion protein was purified from

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107 multimers by ion exchange chromatography on S-Sepharose {Pharmacia, Sweden): The soluble fraction obtained after refolding was gel filtered on Sephadex G-25 (Pharmacia, Sweden) into a buffer containing 5 mM NaCl and 5 mM Tris-HCl pH 8, diluted to double volume with water and then applied to the S-Sepharose column, which was then eluted using a gradient (5 column volumes from 2.5 mM Tris-HCl pH 8, 2.5 mM NaCl to 25 mM Tris-HCl pH 8, 100 mM NaCl). The monomeric correctly folded fusion protein purified to >95% purity (Fig. 32, lanes

6 and 7) was then made 6 M in guanidinium hydrochloride and

0.1 M in DTE, gel filtrated into a buffer containing 8 M urea, 50 mM Tris-HCl pH 8, 1 M NaCl and 10 mM 2-mercaptoethanol and then divided into aliquots to be used as starting material for the refolding experiments described below.

Cyclic refolding of purified fusion protein:

An aliquot of denatured reduced fusion protein was applied to a Ni⁺⁺ -loaded NTA column which was then washed with one column volume of a buffer containing 6 M guanidinium hydrochloride, 50 mM Tris-HCl pH 8 and 10 mM 2-mercaptoetha20 nol.

The fusion protein was then subjected to buffer cycling according to the scheme shown in Table 1 using Buffer A: 50 mM Tris-HCl pH 8, 0.5 M NaCl and 3.2 mM/0.4 mM reduced/oxidized glutathione and Buffer B: 50 mM Tris-HCl pH

8, 0.5 M NaCl, 6 M urea and 4 mM reduced glutathione. After completion of buffer cycling the fusion protein was recovered quantitatively in a soluble form by elution of the column with a buffer containing 50 mM Tris-HCl pH 8, 0.5 M NaCl and 20 mM EDTA. 87% was obtained in the correct monomeric disulphide-bridged form (Fig. 32 lanes 8 and 9).

Refolding of purified fusion protein by linear gradient:

An aliquot of denatured reduced fusion protein was applied to a Ni⁺⁺ -loaded NTA column which was then washed with one

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A 24 hour linear gradient from 100% B to 100% A was then applied at 2 ml/min, using Buffer A: 50 mM Tris-HCl pH 8, 0.5 M NaCl and 3.2 mM/0.4 mM reduced/oxidized glutathione and Buffer B: 50 mM Tris-HCl pH 8, 0.5 M NaCl, 6 M urea and 4 mM reduced glutathione. After completion of the gradient the soluble fraction of fusion protein was eluted in a buffer containing 50 mM Tris-HCl pH 8, 0.5 M NaCl and 20 mM EDTA.

The remaining insoluble fraction was extracted from column in a buffer containing 50 mM Tris-HCl pH 8, 1 M NaCl, 8 M urea,

10 mM 2-mercaptoethanol and 20 mM EDTA.

48% of the fusion protein was recovered in the soluble fraction and 60% of the soluble fraction was recovered in the correct monomeric disulphide-bridged form. The overall efficiency of folding obtained was therefore 29% (Fig 33, lanes

5-7).

Refolding of purified fusion protein by buffer step:

An aliquot of denatured reduced fusion protein was applied to a Ni⁺⁺ -loaded NTA column which was then washed with one column volume of a buffer containing 6 M guanidinium hydrochloride, 50 mM Tris-HCl pH 8 and 10 mM 2-mercaptoethanol .

Buffer containing 50 mM Tris-HCl pH 8, 0.5 M NaCl and

3.2 mM/0.4 mM reduced/oxidized glutathione was then applied to the column at 2 ml/min for 24 hours before recovering the soluble fraction of fusion protein in a buffer containing 50 mM Tris-HCl pH 8, 0.5 M NaCl and 20 mM EDTA. The remaining insoluble fraction was extracted from column in a buffer

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34% of the fusion protein was recovered in the soluble fraction and 28% of the soluble fraction was recovered in the correct monomeric disulphide-bridged form. The overall efficiency of folding obtained was therefore 9.5% (Fig 33, lanes 1-3) .

Conclusions

In summary, using human β₂-microglobulin as a model protein, 10 it may be concluded that (a) straightforward buffer optimization and improved purification of fusion protein prior to cyclic refolding increased refolding yield significantly (from 53% to 87%) and (b) progressive dénaturation renaturation cycling is superior to single-pass refolding under otherwise comparable experimental conditions by a very large factor (87% versus 29% or 9.5% yields).

REFERENCES :

Christensen, J.H. , Hansen, P.K., Lillelund, 0., and Thogersen, H.C. (1991). Sequence-specific binding of the N-terminal three-finger fragment of Xenopus transcription factor IIIA to the internal control region of a 5S RNA gene. FEBS Letters, 295: 181 - 184.

Dalboge, H., Dahl, H.-H., M., Pedersen, J., Hansen, J., W., and T., Kristensen (1987) . A Novel Enzymatic Method for

Production of Authentic hGH From an Eschericia coli Produced hGh-Precursor. Bio/Technology, 5: 161-164.

Datar, R., V., Cartwright, T., and C.-G. Rosen (1993). Process Economics of Animal Cell and Bacterial Fermentations: A

Case Study Analysis of Tissue Plasminogen Activator.

Bio/Technology, 11: 349 - 357.

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Herz, J., Hanmann, U. , Rogne, S., Myklebost, 0., Gausepohl,

H. , and Stanley, K.K. (1988), Surface location and high affinity for calcium of a 500 kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J., 7: 4119-4127.

Hoffmann, H. J., Olsen, E., Etzerodt, M. , Madsen, P., Thagersen, H. C., Kruse, T., and Celis J. E. (1994). Psoriasin Binds Calcium and Is Differentially Regulated With Respect to Other Members of the S100 Protein Family. J. Dermatol.

Invest. In press.

Hochuli, E., W. Bannwarth, H. Dôbeli, R. Gentz, and D. Stüber. 1988. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent.

Bio/Technology, 6: 1321-1325.

Holliger., P., Prospero, T., and G. Winter (1993). Diabodies: Small bivalent and bispecific antibody fragments.

Proc. Natl. Acad. Sci. USA. 90: 6444 - 6448.

Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor,

N.Y.

Nagai, K., and H.C. Thagersen. 1987. Synthesis and Sequence-Specific Proteolysis of Hybrid Proteins Produced in Escherichia coli. Methods in Enzymology, 152: 461-481.

Nagai, K., Nakaseko, Y., Nasmyth, K., and Rhodes, D. (1988).

Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA. Nature, 332: 284 - 286.

Nykjær A., Petersen C.M., Mailer B., Jensen P.H., Moestrup S.K., Holtet T.L., Etzerodt M., Thagersen H.C., Munch M., Andreasen P.A., and Gliemann J. (1992). Purified a₂-Macro30 globulin Receptor/LDL Receptor-related Protein Binds Uroki2155335

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111 nase-Plasminogen Activator Inhibitor Type-1 Complex. J. Biol Chem. 267: 14543-14546.

Rathjen, D. et al. (1991), Mol. Immunol. 28, p29.

Rathjen, D. et al. (1992), Brit. J. Cancer 65, 852-Θ56.

Saiki, R.K., Gelfant, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487 -491.

Studier, F.W. and Moffat, B.A. 1986. Use of Bacteriophage T7 10 RNA Polymerase to Direct Selective High-level Expression of

Cloned Genes. J. Mol. Biol., 189: 113 - 130.

The Regents of the University of California. Enterokinase-cleavable linker sequence. EP 035384.

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SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME : Denzyme ApS (B) STREET: Gustav Wieds Vej 10 (C) CITY: Aarhus C (E) COUNTRY: Denmark (F) POSTAL CODE (ZIP): 0000 (ii) TITLE OF INVENTION: Improved method for the refolding of proteins (iii) NUMBER OF SEQUENCES: 47 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 1554 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Bos taurus (ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 76..1551 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

AGCCTGGGCG AGCGGACCTT GCCCTGGAGG

CGGAAGGGCC CCACC ATG GCG GGC CTG

Met Ala Gly Leu 1

CCTGTTGCGG CAGGGACTCA CGGCTGTCCT

CTG CAT CTC GTT CTG CTC AGC ACC Leu His Leu Val Leu Leu Ser Thr

10

111

<td rowspan="2"> GCC Ala</td><td colspan="2"> CTG GGC</td><td rowspan="2"> GGC Gly</td><td colspan="2"> CTC CTG</td><td colspan="2"> CGG CCG</td><td colspan="2"> GCG GGG</td><td rowspan="2"> AGC Ser</td><td colspan="2"> GTG TTC</td><td rowspan="2"> CTG Leu</td><td rowspan="2"> CCC Pro</td><td rowspan="2"> CGG Arg</td><td rowspan="2"> 159</td>

<td> Leu</td><td> Gly 15</td><td> Leu</td><td> Leu</td><td> Arg</td><td> Pro 20</td><td> Ala</td><td> Gly</td><td> Val</td><td> Phe 25</td>

<td> GAC</td><td> CAG</td><td> GCC</td><td> CAC</td><td> CGT</td><td> GTC</td><td> CTG</td><td> CAG</td><td> AGA</td><td> GCC</td><td> CGC</td><td> AGG</td><td> GCC</td><td> AAC</td><td> TCA</td><td> TTC</td><td> 207</td>

<td> Asp</td><td> Gin</td><td> Ala</td><td> His</td><td> Arg</td><td> Val</td><td> Leu</td><td> Gin</td><td> Arg</td><td> Ala</td><td> Arg</td><td> Arg</td><td> Ala</td><td> Asn</td><td> Ser</td><td> Phe</td><td></td>

35 40

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<td colspan="2"> TTG GAG</td><td colspan="2" rowspan="2"> GAG GTG Glu Val</td><td colspan="12"> AAG CAG GGA AAC CTG GAG CGA GAG TGC CTG GAG GAG</td><td rowspan="2"> 255</td>

<td> Leu 45</td><td> Glu</td><td> Lys</td><td colspan="2"> Gin Gly 50</td><td> Asn</td><td> Leu</td><td> Glu</td><td> Arg 55</td><td> Glu</td><td> Cys</td><td> Leu</td><td> Glu</td><td> Glu 60</td>

<td> GCC</td><td> TGC</td><td> TCA</td><td> CTA</td><td> GAG</td><td> GAG</td><td> GCC</td><td> CGC</td><td> GAG</td><td> GTC</td><td> TTC</td><td> GAG</td><td> GAC</td><td> GCA</td><td> GAG</td><td> CAG</td><td> 303</td>

<td> Al a</td><td> Cys</td><td> Ser</td><td> Leu</td><td> Glu</td><td> Glu</td><td> Ala</td><td> Arg</td><td> Glu</td><td> Val</td><td> Phe</td><td> Glu</td><td> Asp</td><td> Ala</td><td> Glu</td><td> Gin</td><td></td>

<td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td>

<td> ACG</td><td> GAT</td><td> GAA</td><td> TTC</td><td> TGG</td><td> AGT</td><td> AAA</td><td> TAC</td><td> AAA</td><td> GAT</td><td> GGA</td><td> GAC</td><td> CAG</td><td> TGT</td><td> GAA</td><td> GGC</td><td> 351</td>

<td> Thr</td><td> Asp</td><td> Glu</td><td> Phe</td><td> Trp</td><td> Ser</td><td> Lys</td><td> æy_r</td><td> Lys</td><td> Asp</td><td> Gly</td><td> Asp</td><td> Gin</td><td> Cys</td><td> Glu</td><td> Gly</td><td></td>

<td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td>

<td> CAC</td><td> CCG</td><td> TGC</td><td> CTG</td><td> AAT</td><td> CAG</td><td> GGC</td><td> CAC</td><td> TGT</td><td> AAA</td><td> GAC</td><td> GGC</td><td> ATC</td><td> GGA</td><td> GAC</td><td> TAC</td><td> 399</td>

<td> His</td><td> Pro</td><td> Cys</td><td> Leu</td><td> Asn</td><td> Gin</td><td> Gly</td><td> His</td><td> Cys</td><td> Lys</td><td> Asp</td><td> Giy</td><td> Ile</td><td> Gly</td><td> Asp</td><td> Tyr</td><td></td>

<td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td>

<td> ACC</td><td> TGC</td><td> ACC</td><td> TGT</td><td> GCG</td><td> GAA</td><td> GGG</td><td> TTT</td><td> GAA</td><td> GGC</td><td> AAA</td><td> AAC</td><td> TGC</td><td> GAG</td><td> TTC</td><td> TCC</td><td> 447</td>

<td> Thr</td><td> Cys</td><td> Thr</td><td> Cys</td><td> Ala</td><td> Glu</td><td> Gly</td><td> Phe</td><td> Glu</td><td> Gly</td><td> Lys</td><td> Asn</td><td> Cys</td><td> Glu</td><td> Phe</td><td> Ser</td><td></td>

<td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td></td>

<td> ACG</td><td> CGT</td><td> GAG</td><td> ATC</td><td> TGC</td><td> AGC</td><td> CTG</td><td> GAC</td><td> AAT</td><td> GGA</td><td> GGC</td><td> TGC</td><td> GAC</td><td> CAG</td><td> TTC</td><td> TGC</td><td> 495</td>

<td> Thr</td><td> Arg</td><td> Glu</td><td> Ile</td><td> Cys</td><td> Ser</td><td> Leu</td><td> Asp</td><td> Asn</td><td> Gly</td><td> Gly</td><td> Cys</td><td> Asp</td><td> Gin</td><td> Phe</td><td> Cys</td><td></td>

<td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td>

<td> AGG</td><td> GAG</td><td> GAG</td><td> CGC</td><td> AGC</td><td> GAG</td><td> GTG</td><td> CGG</td><td> TGC</td><td> TCC</td><td> TGC</td><td> GCG</td><td> CAC</td><td> GGC</td><td> TAC</td><td> GTG</td><td> 543</td>

<td> Arg</td><td> Glu</td><td> Glu</td><td> Arg</td><td> Ser</td><td> Glu</td><td> Val</td><td> Arg</td><td> Cys</td><td> Ser</td><td> Cys</td><td> Ala</td><td> His</td><td> Gly</td><td> Tyr</td><td> Val</td><td></td>

<td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td>

<td> CTG</td><td> GGC</td><td> GAC</td><td> GAC</td><td> AGC</td><td> AAG</td><td> TCC</td><td> TGC</td><td> GTG</td><td> TCC</td><td> ACA</td><td> GAG</td><td> CGC</td><td> TTC</td><td> CCC</td><td> TGT</td><td> 591</td>

<td> Leu</td><td colspan="2"> Gly Asp</td><td> Asp</td><td> Ser</td><td> Lys</td><td> Ser</td><td> Cys</td><td> Val</td><td> Ser</td><td> Thr</td><td> Glu</td><td> Arg</td><td> Phe</td><td> Pro</td><td> cys</td><td></td>

<td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td>

<td> GGG</td><td> AAG</td><td> TTC</td><td> ACG</td><td> CAG</td><td> GGA</td><td> CGC</td><td> AGC</td><td> CGG</td><td> CGG</td><td> TGG</td><td> GCC</td><td> ATC</td><td> CAC</td><td> ACC</td><td> AGC</td><td> 639</td>

<td> Gly</td><td> Lys</td><td> Phe</td><td> Thr</td><td> Gin</td><td> Gly</td><td> Arg.</td><td> Ser</td><td> Arg</td><td> Arg</td><td> Trp</td><td> Ala</td><td> Ile</td><td> His</td><td> Thr</td><td> Ser</td><td></td>

<td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td>

<td> GAG</td><td> GAC</td><td> GCG</td><td> CTT</td><td> GAC</td><td> GCC</td><td> AGC</td><td> GAG</td><td> CTG</td><td> GAG</td><td> CAC</td><td> TAC</td><td> GAC</td><td> CCT</td><td> GCA</td><td> GAC</td><td> 687</td>

<td> Glu</td><td> Asp</td><td> Ala</td><td> Leu</td><td> Asp</td><td> Ala</td><td> Ser</td><td> Glu</td><td> Leu</td><td> Glu</td><td> His</td><td> Tyr</td><td> Asp</td><td> Pro</td><td> Ala</td><td> Asp</td><td></td>

<td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td></td>

<td> CTG</td><td> AGC</td><td> CCC</td><td> ACA</td><td> GAG</td><td> AGC</td><td> TCC</td><td> TTG</td><td> GAC</td><td> CTG</td><td> CTG</td><td> GGC</td><td> CTC</td><td> AAC</td><td> AGG</td><td> ACC</td><td> 735</td>

<td> Leu</td><td> Ser</td><td> Pro</td><td> Thr</td><td> Glu</td><td> Ser</td><td> Ser</td><td> Leu</td><td> Asp</td><td> Leu</td><td> Leu</td><td> Gly</td><td> Leu</td><td> Asn</td><td> Arg</td><td> Thr</td><td></td>

<td> 205</td><td></td><td></td><td></td><td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td>

<td> GAG</td><td> CCC</td><td> AGC</td><td> GCC</td><td> GGG</td><td> GAG</td><td> GAC</td><td> GGC</td><td> AGC</td><td> CAG</td><td> GTG</td><td> GTC</td><td> CGG</td><td> ATA</td><td> GTG</td><td> GGC</td><td> 783</td>

<td> Glu</td><td> Pro</td><td> Ser</td><td> Ala</td><td> Gly</td><td> Glu</td><td> Asp</td><td> Gly</td><td> Ser</td><td> Gin</td><td> Val</td><td> Val</td><td> Arg</td><td> Ile</td><td> Val</td><td> Gly</td><td></td>

<td></td><td></td><td></td><td></td><td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td>

<td> GGC</td><td> AGG</td><td> GAC</td><td> TGC</td><td> GCG</td><td> GAG</td><td> GGC</td><td> GAG</td><td> TGC</td><td> CCA</td><td> TGG</td><td> CAG</td><td> GCT</td><td> CTG</td><td> CTG</td><td> GTC</td><td> 831</td>

<td> Gly</td><td> Arg</td><td> Asp</td><td> Cys</td><td> Ala</td><td> Glu</td><td> Gly</td><td> Glu</td><td> Cys</td><td> Pro</td><td> Trp</td><td> Gin</td><td> Ala</td><td> Leu</td><td> Leu</td><td> Val</td><td></td>

<td></td><td></td><td></td><td> 240</td><td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td>

<td> AAC</td><td> GAA</td><td> GAG</td><td> AAC</td><td> GAG</td><td> GGA</td><td> TTC</td><td> TGC</td><td> GGG</td><td> GGC</td><td> ACC</td><td> ATC</td><td> CTG</td><td> AAC</td><td> GAG</td><td> TTC</td><td> 879</td>

<td> Asn</td><td> Glu</td><td> Glu</td><td> Asn</td><td> Glu</td><td> Gly</td><td> Phe</td><td> Cys</td><td> Gly</td><td> Gly</td><td> Thr</td><td> Ile</td><td> Leu</td><td> Asn</td><td> Glu</td><td> Phe</td><td></td>

<td></td><td></td><td> 255</td><td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td>

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PCT/DK94/00054

114

<td colspan="2"> TAC GTC</td><td colspan="7"> CTC ACG GCT GCC CAC TGC CTG</td><td rowspan="2"> CAC His</td><td colspan="3"> CAG GCC AAG</td><td rowspan="2"> AGG Arg</td><td rowspan="2"> TTC Phe</td><td rowspan="2"> ACG Thr</td><td rowspan="2"> 927</td>

<td> Tyr</td><td> Val 270</td><td> Leu</td><td> Thr</td><td colspan="2"> Ala Ala</td><td> His 275</td><td> Cys</td><td> Leu</td><td> Gin</td><td> Ala 280</td><td> Lys</td>

<td> GTG</td><td> AGG</td><td> GTC</td><td> GGC</td><td> GAC</td><td> CGG</td><td> AAC</td><td> ACA</td><td> GAG</td><td> CAG</td><td> GAG</td><td> GAG</td><td> GGC</td><td> AAC</td><td> GAG</td><td> ATG</td><td> 975</td>

<td> Val</td><td> Arg</td><td> Val</td><td> Gly</td><td> Asp</td><td> Arg</td><td> Asn</td><td> Thr</td><td> Glu</td><td> Gin</td><td> Glu</td><td> Glu</td><td> Gly</td><td> Asn</td><td> Glu</td><td> Met</td><td></td>

<td> 285</td><td></td><td></td><td></td><td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td>

<td> GCA</td><td> CAC</td><td> GAG</td><td> GTG</td><td> GAG</td><td> ATG</td><td> ACT</td><td> GTG</td><td> AAG</td><td> CAC</td><td> AGC</td><td> CGC</td><td> TTT</td><td> GTC</td><td> AAG</td><td> GAG</td><td> 1023</td>

<td> Ala</td><td> His</td><td> Glu</td><td> Val</td><td> Glu</td><td> Met</td><td> Thr</td><td> Val</td><td> Lys</td><td> His</td><td> Ser</td><td> Arg</td><td> Phe</td><td> Val</td><td> Lys</td><td> Glu</td><td></td>

<td></td><td></td><td></td><td></td><td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td>

<td> ACC</td><td> TAC</td><td> GAC</td><td> TTC</td><td> GAC</td><td> ATC</td><td> GCG</td><td> GTG</td><td> CTG</td><td> AGG</td><td> CTC</td><td> AAG</td><td> ACG</td><td> CCC</td><td> ATC</td><td> CGG</td><td> 1071</td>

<td> Thr</td><td> Tyr</td><td> Asp</td><td> Phe</td><td> Asp</td><td> lie</td><td> Ala</td><td> Val</td><td> Leu</td><td> Arg</td><td> Leu</td><td> Lys</td><td> Thr</td><td> Pro</td><td> He</td><td> Arg</td><td></td>

<td></td><td></td><td></td><td> 320</td><td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td>

<td> TTC</td><td> CGC</td><td> CGG</td><td> AAC</td><td> GTG</td><td> GCG</td><td> CCC</td><td> GCC</td><td> TGC</td><td> CTG</td><td> CCC</td><td> GAG</td><td> AAG</td><td> GAC</td><td> TGG</td><td> GCG</td><td> 1119</td>

<td> Phe</td><td> Arg</td><td> Arg</td><td> Asn</td><td> Val</td><td> Ala</td><td> Pro</td><td> Ala</td><td> Cys</td><td> Leu</td><td> Pro</td><td> Glu</td><td> Lys</td><td> Asp</td><td> Trp</td><td> Ala</td><td></td>

<td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td>

<td> GAG</td><td> GCC</td><td> ACG</td><td> CTG</td><td> ATG</td><td> ACC</td><td> CAG</td><td> AAG</td><td> ACG</td><td> GGC</td><td> ATC</td><td> GTC</td><td> AGC</td><td> GGC</td><td> TTC</td><td> GGG</td><td> 1167</td>

<td> Glu</td><td> Ala</td><td> Thr</td><td> Leu</td><td> Met</td><td> Thr</td><td> Gin</td><td> Lys</td><td> Thr</td><td> Gly</td><td> He</td><td> Val</td><td> Ser</td><td> Gly</td><td> Phe</td><td> Gly</td><td></td>

<td></td><td> 350</td><td></td><td></td><td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td></td>

<td> CGC</td><td> ACG</td><td> CAC</td><td> GAG</td><td> AAG</td><td> GGC</td><td> CGC</td><td> CTG</td><td> TCG</td><td> TCC</td><td> ACG</td><td> CTC</td><td> AAG</td><td> ATG</td><td> CTG</td><td> GAG</td><td> 1215</td>

<td> Arg</td><td> Thr</td><td> His</td><td> Glu</td><td> Lys</td><td> Gly</td><td> Arg</td><td> Leu</td><td> Ser</td><td> Ser</td><td> Thr</td><td> Leu</td><td> Lys</td><td> Met</td><td> Leu</td><td> Glu</td><td></td>

<td> 365</td><td></td><td></td><td></td><td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td>

<td> GTG</td><td> CCC</td><td> TAC</td><td> GTG</td><td> GAC</td><td> CGC</td><td> AGC</td><td> ACC</td><td> TGT</td><td> AAG</td><td> CTG</td><td> TCC</td><td> AGC</td><td> AGC</td><td> TTC</td><td> ACC</td><td> 1263</td>

<td> Val</td><td> Pro</td><td> Tyr</td><td> Val</td><td> Asp</td><td> Arg</td><td> Ser</td><td> Thr</td><td> Cys</td><td> Lys</td><td> Leu</td><td> Ser</td><td> Ser</td><td> Ser</td><td> Phe</td><td> Thr</td><td></td>

<td></td><td></td><td></td><td></td><td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td>

<td> ATT</td><td> ACG</td><td> CCC</td><td> AAC</td><td> ATG</td><td> TTC</td><td> TGC</td><td> GCC</td><td> GGC</td><td> TAC</td><td> GAC</td><td> ACC</td><td> CAG</td><td> CCC</td><td> GAG</td><td> GAC</td><td> 1311</td>

<td> He</td><td> Thr</td><td> Pro</td><td> Asn</td><td> Met</td><td> Phe</td><td> Cys</td><td> Ala</td><td> Gly</td><td> Tyr</td><td> Asp</td><td> Thr</td><td> Gin</td><td> Pro</td><td> Glu</td><td> Asp</td><td></td>

<td></td><td></td><td></td><td> 400</td><td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td>

<td rowspan="2"> GCC Ala</td><td colspan="4"> TGC CAG GGC GAC</td><td colspan="4"> AGT GGC GGC CCC</td><td colspan="3"> CAC GTC ACC</td><td colspan="3"> CGC TTC AAG</td><td rowspan="2"> GAC Asp</td><td rowspan="2"> 1359</td>

<td colspan="2"> Cys Gin 415</td><td> Gly</td><td> Asp</td><td> Ser</td><td> Gly</td><td> Gly 420</td><td> Pro</td><td> His</td><td> Val</td><td> Thr</td><td colspan="2"> Arg Phe 425</td><td> Lys</td>

<td> ACC</td><td> TAC</td><td> TTC</td><td> GTC</td><td> ACA</td><td> GGC</td><td> ATC</td><td> GTC</td><td> AGC</td><td> TGG</td><td> GGA</td><td> GAA</td><td> GGG</td><td> TGC</td><td> GCG</td><td> CGC</td><td> 1407</td>

<td> Thr</td><td> Tyr</td><td> Phe</td><td> Val</td><td> Thr</td><td> Gly</td><td> He</td><td> Val</td><td> Ser</td><td> Trp</td><td> Gly</td><td> Glu</td><td> Gly</td><td> Cys</td><td> Ala</td><td> Arg</td><td></td>

<td></td><td> 430</td><td></td><td></td><td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td></td>

<td> AAG</td><td> GGC</td><td> AAG</td><td> TTC</td><td> GGC</td><td> GTC</td><td> TAC</td><td> ACC</td><td> AAG</td><td> GTC</td><td> TCC</td><td> AAC</td><td> TTC</td><td> CTC</td><td> AAG</td><td> TGG</td><td> 1455</td>

<td> Lys</td><td> Gly</td><td> Lys</td><td> Phe</td><td> Gly</td><td> Val</td><td> Tyr</td><td> Thr</td><td> Lys</td><td> Val</td><td> Ser</td><td> Asn</td><td> Phe</td><td> Leu</td><td> Lys</td><td> Trp</td><td></td>

<td> 445</td><td></td><td></td><td></td><td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td>

<td> ATC</td><td> GAC</td><td> AAG</td><td> ATC</td><td> ATG</td><td> AAG</td><td> GCC</td><td> AGG</td><td> GCA</td><td> GGG</td><td> GCC</td><td> GCG</td><td> GGC</td><td> AGC</td><td> CGC</td><td> GGC</td><td> 1503</td>

<td> He</td><td> Asp</td><td> Lys</td><td> He</td><td> Met</td><td> Lys</td><td> Ala</td><td> Arg</td><td> Ala</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Gly</td><td> Ser</td><td> Arg</td><td> Gly</td><td></td>

<td></td><td></td><td></td><td></td><td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td>

<td> CAC</td><td> AGT</td><td> GAA</td><td> GCC</td><td> CCT</td><td> GCC</td><td> ACC</td><td> TGG</td><td> ACG</td><td> GTC</td><td> CCG</td><td> CCG</td><td> CCC</td><td> CTC</td><td> CCC</td><td> CTC</td><td> 1551</td>

<td> His</td><td> Ser</td><td> Glu</td><td> Ala</td><td> Pro</td><td> Ala</td><td> Thr</td><td> Trp</td><td> Thr</td><td> Val</td><td> Pro</td><td> Pro</td><td> Pro</td><td> Leu</td><td> Pro</td><td> Leu</td><td></td>

<td></td><td></td><td></td><td> 480</td><td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td>

ΤΑΑ

1554

-MO 94/18227 ? 1 ί i '3 ? h

H# X V 3 ·) Ô 3

115 (2) INFORMATION FOR SEQ ID NO : 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 492 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

PCT/DK94/00054 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

<td> Met 1</td><td> Ala</td><td> Gly</td><td> Leu</td><td> Leu 5</td><td> His</td><td> Leu</td><td> Val</td><td> Leu</td><td> Leu 10</td><td> Ser</td><td> Thr</td><td> Ala</td><td> Leu</td><td> Gly 15</td><td> Gly</td>

<td> Leu</td><td> Leu</td><td> Arg</td><td> Pro 20</td><td> Ala</td><td> Gly</td><td> Ser</td><td> Val</td><td> Phe 25</td><td> Leu</td><td> Pro</td><td> Arg</td><td> Asp</td><td> Gin 30</td><td> Ala</td><td> His</td>

<td> Arg</td><td> Val</td><td> Leu 35</td><td> Gin</td><td> Arg</td><td> Ala</td><td> Arg</td><td> Arg 40</td><td> Ala</td><td> Asn</td><td> Ser</td><td> Phe</td><td> Leu 45</td><td> Glu</td><td> Glu</td><td> Val</td>

<td> Lys</td><td> Gin 50</td><td> Gly</td><td> Asn</td><td> Leu</td><td> Glu</td><td> Arg 55</td><td> Glu</td><td> Cys</td><td> Leu</td><td> Glu</td><td> Glu 60</td><td> Ala</td><td> Cys</td><td> Ser</td><td> Leu</td>

<td> Glu 65</td><td> Glu</td><td> Ala</td><td> Arg</td><td> Glu</td><td> Val 70</td><td> Phe</td><td> Glu</td><td> Asp</td><td> Ala</td><td> Glu 75</td><td> Gin</td><td> Thr</td><td> Asp</td><td> Glu</td><td> Phe 80</td>

<td> Trp</td><td> Ser</td><td> Lys</td><td> Tyr</td><td> Lys 85</td><td> Asp</td><td> Gly</td><td> Asp</td><td> Gin</td><td> Cys 90</td><td> Glu</td><td> Gly</td><td> His</td><td> Pro</td><td> Cys 95</td><td> Leu</td>

<td> Asn</td><td> Gin</td><td> Gly</td><td> His 100</td><td> Cys</td><td> Lys</td><td> Asp</td><td> Gly</td><td> Ile 105</td><td> Gly</td><td> Asp</td><td> Tyr</td><td> Thr</td><td> Cys 110</td><td> Thr</td><td> Cys</td>

<td> Ala</td><td> Glu</td><td> Gly 115</td><td> Phe</td><td> Glu</td><td> Gly</td><td> Lys</td><td> Asn 120</td><td> Cys</td><td> Glu</td><td> Phe</td><td> Ser</td><td> Thr 125</td><td> Arg</td><td> Glu</td><td> Ile</td>

<td> Cys</td><td> Ser 130</td><td> Leu</td><td> Asp</td><td> Asn</td><td> Gly</td><td> Gly 135</td><td> Cys</td><td> Asp</td><td> Gin</td><td> Phe</td><td> Cys 140</td><td> Arg</td><td> Glu</td><td> Glu</td><td> Arg</td>

<td> Ser 145</td><td> Glu</td><td> Val</td><td> Arg</td><td> Cys</td><td> Ser 150</td><td> Cys</td><td> Ala</td><td> His</td><td> Gly</td><td> Tyr 155</td><td> Val</td><td> Leu</td><td> Gly</td><td> Asp</td><td> Asp 160</td>

<td> Ser</td><td> Lys</td><td> Ser</td><td> Cys</td><td> Val 165</td><td> Ser</td><td> Thr</td><td> Glu</td><td> Arg</td><td> Phe 170</td><td> Pro</td><td> Cys</td><td> Gly</td><td> Lys</td><td> Phe 175</td><td> Thr</td>

<td> Gin</td><td> Gly</td><td> Arg</td><td> Ser 180</td><td> Arg</td><td> Arg</td><td> Trp</td><td> Ala</td><td> Ile 185</td><td> His</td><td> Thr</td><td> Ser</td><td> Glu</td><td> Asp 190</td><td> Ala</td><td> Leu</td>

<td> Asp</td><td> Ala</td><td> Ser 195</td><td> Glu</td><td> Leu</td><td> Glu</td><td> His</td><td> Tyr 200</td><td> Asp</td><td> Pro</td><td> Ala</td><td> Asp</td><td> Leu 205</td><td> Ser</td><td> Pro</td><td> Thr</td>

<td> Glu</td><td> Ser 210</td><td> Ser</td><td> Leu</td><td> Asp</td><td> Leu</td><td> Leu 215</td><td> Gly</td><td> Leu</td><td> Asn</td><td> Arg</td><td> Thr 220</td><td> Glu</td><td> Pro</td><td> Ser</td><td> Ala</td>

<td> Gly 225</td><td> Glu</td><td> Asp</td><td> Gly</td><td> Ser</td><td> Gin 230</td><td> Val</td><td> Val</td><td> Arg</td><td> Ile</td><td> Val 235</td><td> Gly</td><td> Gly</td><td> Arg</td><td> Asp</td><td> Cys 240</td>

Ala Glu Gly Glu Cys Pro Trp Gin Ala Leu Leu Val Asn Glu Glu Asn 245 250 255

WO 94/18227

PCT/DK94/00054 2155335

<td rowspan="2"> Glu</td><td colspan="2" rowspan="2"> Gly Phe</td><td colspan="3" rowspan="2"> Cys Gly Gly 260</td><td rowspan="2"> Thr</td><td rowspan="2"> He</td><td colspan="6"> Leu Asn Glu Phe Tyr Val</td><td rowspan="2"> Leu</td><td rowspan="2"> Thr</td>

<td colspan="3"> 265</td><td colspan="3"> 270</td>

<td> Ala</td><td> Ala</td><td> His</td><td> Cys</td><td> Leu</td><td> His</td><td> Gin</td><td> Ala</td><td> Lys</td><td> Arg</td><td> Phe</td><td> Thr</td><td> Val</td><td> Arg</td><td> Val</td><td> Gly</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td> Asp</td><td> Arg</td><td> Asn</td><td> Thr</td><td> Glu</td><td> Gin</td><td> Glu</td><td> Glu</td><td> Gly</td><td> Asn</td><td> Glu</td><td> Met</td><td> Ala</td><td> His</td><td> Glu</td><td> Val</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td> Glu</td><td> Met</td><td> Thr</td><td> Val</td><td> Lys</td><td> His</td><td> Ser</td><td> Arg</td><td> Phe</td><td> Val</td><td> Lys</td><td> Glu</td><td> Thr</td><td> Tyr</td><td> Asp</td><td> Phe</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td> Asp</td><td> lie</td><td> Ala</td><td> Val</td><td> Leu</td><td> Arg</td><td> Leu</td><td> Lys</td><td> Thr</td><td> Pro</td><td> He</td><td> Arg</td><td> Phe</td><td> Arg</td><td> Arg</td><td> Asn</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td> Val</td><td> Ala</td><td> Pro</td><td> Ala</td><td> Cys</td><td> Leu</td><td> Pro</td><td> Glu</td><td> Lys</td><td> Asp</td><td> Trp</td><td> Ala</td><td> Glu</td><td> Ala</td><td> Thr</td><td> Leu</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td> Met</td><td> Thr</td><td> Gin</td><td> Lys</td><td> Thr</td><td> Gly</td><td> He</td><td> Val</td><td> Ser</td><td> Gly</td><td> Phe</td><td> Gly</td><td> Arg</td><td> Thr</td><td> His</td><td> Glu</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td> Lys</td><td> Gly</td><td> Arg</td><td> Leu</td><td> Ser</td><td> Ser</td><td> Thr</td><td> Leu</td><td> Lys</td><td> Met</td><td> Leu</td><td> Glu</td><td> Val</td><td> Pro</td><td> Tyr</td><td> Val</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td> Asp</td><td> Arg</td><td> Ser</td><td> Thr</td><td> Cys</td><td> Lys</td><td> Leu</td><td> Ser</td><td> Ser</td><td> Ser</td><td> Phe</td><td> Thr</td><td> He</td><td> Thr</td><td> Pro</td><td> Asn</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td> Met</td><td> Phe</td><td> Cys</td><td> Ala</td><td> Gly</td><td> Tyr</td><td> Asp</td><td> Thr</td><td> Gin</td><td> Pro</td><td> Glu</td><td> Asp</td><td> Ala</td><td> Cys</td><td> Gin</td><td> Gly</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td> Asp</td><td> Ser</td><td> Gly</td><td> Gly</td><td> Pro</td><td> His</td><td> Val</td><td> Thr</td><td> Arg</td><td> Phe</td><td> Lys</td><td> Asp</td><td> Thr</td><td> Tyr</td><td> Phe</td><td> Val</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td> Thr</td><td> Gly</td><td> He</td><td> Val</td><td> Ser</td><td> Trp</td><td> Gly</td><td> Glu</td><td> Gly</td><td> Cys</td><td> Ala</td><td> Arg</td><td> Lys</td><td> Gly</td><td> Lys</td><td> Phe</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td> Gly</td><td> Val</td><td> Tyr</td><td> Thr</td><td> Lys</td><td> Val</td><td> Ser</td><td> Asn</td><td> Phe</td><td> Leu</td><td> Lys</td><td> Trp</td><td> He</td><td> Asp</td><td> Lys</td><td> He</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td> Met</td><td> Lys</td><td> Ala</td><td> Arg</td><td> Ala</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Gly</td><td> Ser</td><td> Arg</td><td> Gly</td><td> His</td><td> Ser</td><td> Glu</td><td> Ala</td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td> Pro</td><td> Ala</td><td> Thr</td><td> Trp</td><td> Thr</td><td> Val</td><td> Pro</td><td> Pro</td><td> Pro</td><td> Leu</td><td> Pro</td><td> Leu</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> (2)</td><td colspan="3"> INFORMATION</td><td> FOR</td><td> SEQ</td><td colspan="2"> ID NO: :</td><td> i :</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

Ci) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs CS) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3:

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117

CGTCCTGGAT CCATCGAGGG TAGAATCCAG CGTACTCCAA AG (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GCGAAGCTTG ATCACATGTC TCG 23 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CGTCCTGGAT CCATCGAGGG TAGAATCCAG AAAACCCCTC AAAT 44 (2) INFORMATION FOR SEQ ID NO : 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GCGAAGCTTA CATGTCTCGA TC 22 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic)

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118 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CCTGGATCCA TCGAGGGTAG GTTCCCAACC ATTCCCTTAT (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CCGAAGCTTA GAAGCCACAG CTGCCC 26 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CGTCCTGGAT CCATCGAGGG TAGGTACTCG CGGGAGAAG 39 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CGACCGAAGC TTCAGAGTTC GTTGTG 26 (2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

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119 (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CGTCCTGGAT CCATCGAGGG TAGGGCTATC GACGCCCCTA AG 42 (2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CGACCGAAGC TTATCGGCAG TGGGGCCCCT 3 0 (2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CGACCGAAGC TTAGGCCTTG CAGGAGCGG 29 (2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

CGACCGAAGC TTACTTCTTG CATGACTTCC CG 32 (2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single

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120 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

CGTCCTGGAT CCATCGAGGG TAGGGGCACC AACAAATGCC GG (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CGACCGAAGC TTAGTCCAGG CTGCGGCAG 29 (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

CGTCCTGGAT CCATCGAGGG TAGGGTGCCT CCACCCCAGT G 41 (2) INFORMATION FOR SEQ ID NO : 18:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

CGACCGAAGC TTACTGGTCG CAGAGCTCG 29 ( i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 46 base pairs (2) INFORMATION FOR SEQ ID NO: 19:

Λ***· ^w0 94/18227

<img file="CA2155335C_D0020.tif" />

PCT/DK94/00054

121 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

CCTTGATCAA TCGAGGGTAG GGGTGGTCAG TGCTCTCTGA ATAACG (2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single &lt;D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

CGCAAGCTTA CTTAAACTCA TAGCAGGTG (2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

CGTCCTGGAT CCATCGAGGG TAGGGCGGTG AATTCCTCTT GCCG (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

CGACCGAAGC TTAGATGTGG CAGCCACGCT (2) INFORMATION FOR SEQ ID NO: 23:

WO 94/18227 .. .« n

3 0 0 3

122 (i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid CC) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

CGTCCTGGAT CCATCGAGGG TAGGGTGTCC AACTGCACGG CT

<img file="CA2155335C_D0021.tif" />

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

CGACCGAAGC TTAGATGCTG CAGTCCTCCT (2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

CGTCCTGGAT CCATCGAGGG TAGGAGTAAA TACAAAGATG GAGACCA (2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

PCT/DK94/00054

CGACCGAAGC TTACCAGGTG GCAGGGGCTT

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123 (2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 base pairs (B) TYPF: nucleic acid (C) STR^T.NDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

CTGCCTGGAT CCATCGAGGG TAGGAAAGTG TATCTCTCAT CAGAGTGCAA GACTGGGAAT GG (2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

CGACCGAAGC TTATTCACAC TCAAGAATGT CGC 33 (2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

CTGCCTGGAT CCATCGAGGG TAGGGTCCAG GACTGCTACC AT 42 (2) INFORMATION FOR SEQ ID NO: 30:

{i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

CGACCGAAGC TTACGCTTCT GTTCCTGAGC A

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124 (2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 31:

CCTGGATCCA TCGAGGGTAG GGTCTACCTC CAGACATCCT 40 (2) INFORMATION FOR SEQ ID NO: 32:

&lt;i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

CCGAAGCTTC AAGCATTTCC AAGATC 26 (2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

CCTGGATCCA TCGAGGGTAG GGGCGAGCCA CCAACCCAG 39 (2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic)

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125 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

CCGAAGCTTA CACGATCCCG AACTG (2) INFORMATION FOR SEQ ID NO : 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

CCGAGATCTA TCGAGGGTAG GCAGGTCAAA CTGCAGCA {2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 36:

GCCAAGCTTA ATTCAGATCC TCTTCTGAG 29 (2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

Gly Ser lie Glu Gly Arg 1 5 (2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

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<img file="CA2155335C_D0022.tif" />

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

Ile Glu Gly Ara 1 (2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

Tyr Trp Thr Asp 1 (2) INFORMATION FOR SEQ ID NO: 40:

( i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

lie Gin Gly Arg 1 (2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

Ala Glu Gly Arg 1

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Μ X.

5533ο

127 (2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

Ala Gin Gly Arg 1 (2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

He Cys Gly Arg 1 (2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

Ala Cys Gly Arg 1 (2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

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PCT/DK94/00054 128 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

lie Met Gly Arg 1 (2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 46:

Ala Met Gly Arg 1 (2) INFORMATION FOR SEQ ID NO : 47:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 6 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

His His His His His His 1 5 (2) INFORMATION FOR SEQ ID NO: 46:

&lt;i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

Met Gly Ser His His His His His His Gly Ser Ile Glu Gly Arg 15 10 15 (2) INFORMATION FOR SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 119 ammo acids

129 (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear

PCT/DK94/00054 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

<td colspan="7"> Met Ser Arg Ser Val Ala Leu</td><td rowspan="2"> Ala</td><td colspan="7"> Val Leu Ala Leu Leu Ser Leu</td><td rowspan="2"> Ser</td>

<td> 1</td><td colspan="6"> 5</td><td colspan="4"> 10</td><td colspan="3"> 15</td>

<td> Gly</td><td> Leu</td><td> Glu</td><td> Ala</td><td> lie</td><td> Gin</td><td> Arg</td><td> Thr</td><td> Pro</td><td> Lys</td><td> He</td><td> Gin</td><td> Val</td><td> Tyr</td><td> Ser</td><td> Arg</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td> His</td><td> Pro</td><td> Ala</td><td> Glu</td><td> Asn</td><td> Gly</td><td> Lys</td><td> Ser</td><td> Asn</td><td> Phe</td><td> Leu</td><td> Asn</td><td> Cys</td><td> Tyr</td><td> Val</td><td> Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td> Gly</td><td> Phe</td><td> His</td><td> Pro</td><td> Ser</td><td> Asp</td><td> He</td><td> Glu</td><td> Val</td><td> Asp</td><td> Leu</td><td> Leu</td><td> Lys</td><td> Asn</td><td> Gly</td><td> Glu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td> Arg</td><td> lie</td><td> Glu</td><td> Lys</td><td> Val</td><td> Glu</td><td> His</td><td> Ser</td><td> Asp</td><td> Leu</td><td> Ser</td><td> Phe</td><td> Ser</td><td> Lys</td><td> Asp</td><td> Trp</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td> Ser</td><td> Phe</td><td> Tyr</td><td> Leu</td><td> Leu</td><td> Tyr</td><td> Tyr</td><td> Thr</td><td> Glu</td><td> Phe</td><td> Thr</td><td> Pro</td><td> Thr</td><td> Glu</td><td> Lys</td><td> Asp</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td> Glu</td><td> Tyr</td><td> Ala</td><td> Cys</td><td> Arg</td><td> Val</td><td> Asn</td><td> His</td><td> Val</td><td> Thr</td><td> Leu</td><td> Ser</td><td> Gin</td><td> Pro</td><td> Lys</td><td> He</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td> Val</td><td> Lys</td><td> Trp</td><td> Asp</td><td> Arg</td><td> Asp</td><td> Met</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

115 (2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 119 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

<td> Met</td><td> Ala</td><td> Arg</td><td> Ser</td><td> Val</td><td> Thr</td><td> Leu</td><td> Val</td><td> Phe</td><td> Leu</td><td> Val</td><td> Leu</td><td> Val</td><td> Ser</td><td> Leu</td><td> Thr</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td> Gly</td><td> Leu</td><td> Tyr</td><td> Ala</td><td> He</td><td> Gin</td><td> Lys</td><td> Thr</td><td> Pro</td><td> Gin</td><td> lie</td><td> Gin</td><td> Val</td><td> Tyr</td><td> Ser</td><td> Arg</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td> His</td><td> Pro</td><td> Pro</td><td> Glu</td><td> Asn</td><td> Gly</td><td> Lys</td><td> Pro</td><td> Asn</td><td> He</td><td> Leu</td><td> Asn</td><td> Cys</td><td> Tyr</td><td> Val</td><td> Thr</td>

Gin Phe His Pro Pro His Ile Glu lie Gin Met Leu Lys Asn Gly Lys 50 55 60

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<img file="CA2155335C_D0024.tif" />

130

<td> Lys 65</td><td> He</td><td> Pro</td><td> Lys</td><td> Val</td><td> Glu 70</td><td> Met</td><td> Ser</td><td> Asp</td><td> Met</td><td> Ser 75</td><td> Phe</td><td> Ser</td><td> Lys</td><td> Asp</td><td> Trp 80</td>

<td> Ser</td><td> Phe</td><td> Tyr</td><td> He</td><td> Leu 85</td><td> Ala</td><td> His</td><td> Thr</td><td> Glu</td><td> Phe 90</td><td> Thr</td><td> Pro</td><td> Thr</td><td> Glu</td><td> Thr 95</td><td> Asp</td>

<td> Thr</td><td> Tyr</td><td> Ala</td><td> Cys 100</td><td> Arg</td><td> Val</td><td> Lys</td><td> His</td><td> Asp 105</td><td> Ser</td><td> Met</td><td> Ala</td><td> Glu</td><td> Pro 110</td><td> Lys</td><td> Thr</td>

<td> Val</td><td> Tyr</td><td> Trp</td><td> Asp</td><td> Arg</td><td> Asp</td><td> Met</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

115 (2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 217 amino acids <B> TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

<td> Met 1</td><td> Ala</td><td> Thr</td><td> Gly</td><td> Ser 5</td><td> Arg</td><td> Thr</td><td> Ser</td><td> Leu</td><td> Leu 10</td><td> Leu</td><td> Ala</td><td> Phe</td><td> Gly</td><td> Leu 15</td><td> Leu</td>

<td> Cys</td><td> Leu</td><td> Pro</td><td> Trp 20</td><td> Leu</td><td> Gin</td><td> Glu</td><td> Gly</td><td> Ser 25</td><td> Ala</td><td> Phe</td><td> Pro</td><td> Thr</td><td> He 30</td><td> Pro</td><td> Leu</td>

<td> Ser</td><td> Arg</td><td> Leu 35</td><td> Phe</td><td> Asp</td><td> Asn</td><td> Ala</td><td> Ser 40</td><td> Leu</td><td> Arg</td><td> Ala</td><td> His</td><td> Arg 45</td><td> Leu</td><td> His</td><td> Gin</td>

<td> Leu</td><td> Ala 50</td><td> Phe</td><td> Asp</td><td> Thr</td><td> Tyr</td><td> Gin 55</td><td> Glu</td><td> Phe</td><td> Glu</td><td> Glu</td><td> Ala 60</td><td> Tyr</td><td> He</td><td> Pro</td><td> Lys</td>

<td> Glu 65</td><td> Gin</td><td> Lys</td><td> Tyr</td><td> Ser</td><td> Phe 70</td><td> Leu</td><td> Gin</td><td> Asn</td><td> Pro</td><td> Gin 75</td><td> Thr</td><td> Ser</td><td> Leu</td><td> Cys</td><td> Phe 80</td>

<td> Ser</td><td> Glu</td><td> Ser</td><td> He</td><td> Pro 85</td><td> Thr</td><td> Pro</td><td> Ser</td><td> Asn</td><td> Arg 90</td><td> Glu</td><td> Glu</td><td> Thr</td><td> Gin</td><td> Gin 95</td><td> Lys</td>

<td> Ser</td><td> Asn</td><td> Leu</td><td> Glu 100</td><td> Leu</td><td> Leu</td><td> Arg</td><td> He</td><td> Ser 105</td><td> Leu</td><td> Leu</td><td> Leu</td><td> He</td><td> Gin 110</td><td> Ser</td><td> Trp</td>

<td> Leu</td><td> Glu</td><td> Pro 115</td><td> Val</td><td> Gin</td><td> Phe</td><td> Leu</td><td> Arg 120</td><td> Ser</td><td> Val</td><td> Phe</td><td> Ala</td><td> Asn 125</td><td> Ser</td><td> Leu</td><td> Val</td>

<td> Tyr</td><td> Gly 130</td><td> Ala</td><td> Ser</td><td> Asp</td><td> Ser</td><td> Asn 135</td><td> Val</td><td> Tyr</td><td> Asp</td><td> Leu</td><td> Leu 140</td><td> Lys</td><td> Asp</td><td> Leu</td><td> Glu</td>

<td> Glu 145</td><td> Gly</td><td> He</td><td> Gin</td><td> Thr</td><td> Leu 150</td><td> Met</td><td> Gly</td><td> Arg</td><td> Leu</td><td> Glu 155</td><td> Asp</td><td> Gly</td><td> Ser</td><td> Pro</td><td> Arg 160</td>

<td> Thr</td><td> Gly</td><td> Gin</td><td> He</td><td> Phe 165</td><td> Lys</td><td> Gin</td><td> Thr</td><td> Tyr</td><td> Ser 170</td><td> Lys</td><td> Phe</td><td> Asp</td><td> Thr</td><td> Asn 175</td><td> Ser</td>

-WO 94/18227

PCT/DK94/00054

131

<td colspan="15"> His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys</td><td rowspan="2"> Phe</td>

<td></td><td colspan="4"> 180</td><td colspan="6"> 185</td><td colspan="4"> 190</td>

<td> Arg</td><td> Lys</td><td> Asp</td><td> Met</td><td> Asp</td><td> Lys</td><td> Val</td><td> Glu</td><td> Thr</td><td> Phe</td><td> Leu</td><td> Arg</td><td> lie</td><td> Val</td><td> Gin</td><td> Cys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td> Arg</td><td> Ser</td><td> Val</td><td> Glu</td><td> Gly</td><td> Ser</td><td> Cys</td><td> Gly</td><td> Phe</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

210 215 (2) INFORMATION FOR SEQ ID NO: 52:

<td rowspan="2"> ii)</td><td colspan="15"> SEQUENCE CHARACTERISTICS: (A) LENGTH: 4544 amino acids</td>

<td> (B) (C) (D)</td><td colspan="14"> TYPE: amino acid STRANDEDNESS : single TOPOLOGY: linear</td>

<td> (ii)</td><td colspan="6"> MOLECULE TYPE: protein</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> (xi)</td><td colspan="8"> SEQUENCE DESCRIPTION: SEQ ID NO:</td><td> : 52 :</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Met 1</td><td> Leu</td><td> Thr</td><td> Pro</td><td> Pro 5</td><td> Leu</td><td> Leu</td><td> Leu</td><td> Leu</td><td> Leu 10</td><td> Pro</td><td> Leu</td><td> Leu</td><td> Ser</td><td> Ala 15</td><td> Leu</td>

<td> Val</td><td> Ala</td><td> Ala</td><td> Ala 20</td><td> He</td><td> Asp</td><td> Ala</td><td> Pro</td><td> Lys 25</td><td> Thr</td><td> Cys</td><td> Ser</td><td> Pro</td><td> Lys 30</td><td> Gin</td><td> Phe</td>

<td> Ala</td><td> Cys</td><td> Arg 35</td><td> Asp</td><td> Gin</td><td> He</td><td> Thr</td><td> Cys 40</td><td> He</td><td> Ser</td><td> Lys</td><td> Gly</td><td> Trp 45</td><td> Arg</td><td> Cys</td><td> Asp</td>

<td> Gly</td><td> Glu 50</td><td> Arg</td><td> Asp</td><td> Cys</td><td> Pro</td><td> Asp 55</td><td> Gly</td><td> Ser</td><td> Asp</td><td> Glu</td><td> Ala £0</td><td> Pro</td><td> Glu</td><td> He</td><td> Cys</td>

<td> Pro 65</td><td> Gin</td><td> Ser</td><td> Lys</td><td> Ala</td><td> Gin 70</td><td> Arg</td><td> Cys</td><td> Gin</td><td> Pro</td><td> Asn 75</td><td> Glu</td><td> His</td><td> Asn</td><td> Cys</td><td> Leu 80</td>

<td> Gly</td><td> Thr</td><td> Glu</td><td> Leu</td><td> Cys B5</td><td> Val</td><td> Pro</td><td> Met</td><td> Ser</td><td> Arg 90</td><td> Leu</td><td> Cys</td><td> Asn</td><td> Gly</td><td> Val 95</td><td> Gin</td>

<td> Asp</td><td> Cys</td><td> Met</td><td> Asp 100</td><td> Gly</td><td> Ser</td><td> Asp</td><td> Glu</td><td> Gly 105</td><td> Pro</td><td> His</td><td> Cys</td><td> Arg</td><td> Glu 110</td><td> Leu</td><td> Gin</td>

<td> Gly</td><td> Asn</td><td> Cys 115</td><td> Ser</td><td> Arg</td><td> Leu</td><td> Gly</td><td> Cys 120</td><td> Gin</td><td> His</td><td> His</td><td> Cys</td><td> Val 125</td><td> Pro</td><td> Thr</td><td> Leu</td>

<td> Asp</td><td> Gly 130</td><td> Pro</td><td> Thr</td><td> Cys</td><td> Tyr</td><td> Cys 135</td><td> Asn</td><td> Ser</td><td> Ser</td><td> Phe</td><td> Gin 140</td><td> Leu</td><td> Gin</td><td> Ala</td><td> Asp</td>

<td> Gly 145</td><td> Lys</td><td> Thr</td><td> Cys</td><td> Lys</td><td> Asp 150</td><td> Phe</td><td> Asp</td><td> Glu</td><td> Cys</td><td> Ser 155</td><td> Val</td><td> Tyr</td><td> Gly</td><td> Thr</td><td> Cys 160</td>

<td> Ser</td><td> Gin</td><td> Leu</td><td> Cys</td><td> Thr 165</td><td> Asn</td><td> Thr</td><td> Asp</td><td> Gly</td><td> Ser 170</td><td> Phe</td><td> He</td><td> Cys</td><td> Gly</td><td> Cys 175</td><td> Val</td>

<td> Glu</td><td> Gly</td><td> Tyr</td><td> Leu 180</td><td> Leu</td><td> Gin</td><td> Pro</td><td> Asp</td><td> Asn 185</td><td> Arg</td><td> Ser</td><td> Cys</td><td> Lys</td><td> Ala 190</td><td> Lys</td><td> Asn</td>

WO 94/18227

<img file="CA2155335C_D0025.tif" />

PCT/DK94/00054

<td> Glu</td><td> Pro</td><td> Val 195</td><td> Asp</td><td> Arg</td><td> Pro</td><td> Pro</td><td> Val 200</td><td> Leu</td><td> Leu</td><td> Ile</td><td> Ala</td><td> Asn 205</td><td> Ser</td><td> Gin</td><td> Asn</td>

<td> Ile</td><td> Leu 210</td><td> Ala</td><td> Thr</td><td> Tyr</td><td> Leu</td><td> Ser 215</td><td> Gly</td><td> Ala</td><td> Gin</td><td> Val</td><td> Ser 220</td><td> Thr</td><td> Ile</td><td> Thr</td><td> Pro</td>

<td> Thr 225</td><td> Ser</td><td> Thr</td><td> Arg</td><td> Gin</td><td> inr 230</td><td> Thr</td><td> Ala</td><td> Met</td><td> Asp</td><td> Phe 235</td><td> Ser</td><td> Tyr</td><td> Ala</td><td> Asn</td><td> Glu 240</td>

<td> Thr</td><td> Val</td><td> Cys</td><td> Trp</td><td> Val 245</td><td> His</td><td> Val</td><td> Gly</td><td> Asp</td><td> Ser 250</td><td> Ala</td><td> Ala</td><td> Gin</td><td> Thr</td><td> Gin 255</td><td> Leu</td>

<td> Lys</td><td> Cys</td><td> Ala</td><td> Arg 260</td><td> Met</td><td> Pro</td><td> Gly</td><td> Leu</td><td> Lys 265</td><td> Gly</td><td> Phe</td><td> Val</td><td> Asp</td><td> Glu 270</td><td> His</td><td> Thr</td>

<td> Ile</td><td> Asn</td><td> Ile 275</td><td> Ser</td><td> Leu</td><td> Ser</td><td> Leu</td><td> His 280</td><td> His</td><td> Val</td><td> Glu</td><td> Gin</td><td> Met 285</td><td> Ala</td><td> Ile</td><td> Asp</td>

<td> Trp</td><td> Leu 290</td><td> Thr</td><td> Gly</td><td> Asn</td><td> Phe</td><td> Tyr 295</td><td> Phe</td><td> Val</td><td> Asp</td><td> Asp</td><td> Ile 300</td><td></td><td> Asp</td><td> Arg</td><td> Ile</td>

<td> Phe 305</td><td> Val</td><td> Cys</td><td> Asn</td><td> Arg</td><td> Asn 310</td><td> Gly</td><td> Asp</td><td> Thr</td><td> Cys</td><td> Val 315</td><td> Thr</td><td> Leu</td><td> Leu</td><td> Asp</td><td> Leu 320</td>

<td> Glu</td><td> Leu</td><td> Tyr</td><td> Asn</td><td> Pro 325</td><td> Lys</td><td> Gly</td><td> Ile</td><td> Ala</td><td> Leu 330</td><td> Asp</td><td> Pro</td><td> Ala</td><td> Met</td><td> Gly 335</td><td> Lys</td>

<td> Val</td><td> Phe</td><td> Phe</td><td> Thr 340</td><td> Asp</td><td> Tyr</td><td> Gly</td><td> Gin</td><td> Ile 345</td><td> Pro</td><td> Lys</td><td> Val</td><td> Glu</td><td> Arg 350</td><td> Cys</td><td> Asp</td>

<td> Met</td><td> Asp</td><td> Gly 355</td><td> Gin</td><td> Asn</td><td> Arg</td><td> Thr</td><td> Lys 360</td><td> Leu</td><td> Val</td><td> Asp</td><td> Ser</td><td> Lys 365</td><td> Ile</td><td> Val</td><td> Phe</td>

<td> Pro</td><td> His 370</td><td> Gly</td><td> Ile</td><td> Thr</td><td> Leu</td><td> Asp 375</td><td> Leu</td><td> Val</td><td> Ser</td><td> Arg</td><td> Leu 380</td><td> Val</td><td> Tyr</td><td> Trp</td><td> Ala</td>

<td> Asp 385</td><td> Ala</td><td> Tyr</td><td> Leu</td><td> Asp</td><td> Tyr 390</td><td> Ile</td><td> Glu</td><td> Val</td><td> Val</td><td> Asp 395</td><td> Tyr</td><td> Glu</td><td> Gly</td><td> Lys</td><td> Gly 400</td>

<td> Arg</td><td> Gin</td><td> Thr</td><td> Ile</td><td> Ile 405</td><td> Gin</td><td> Gly</td><td> Ile</td><td> Leu</td><td> Ile 410</td><td> Glu</td><td> His</td><td> Leu</td><td> Tyr</td><td> Gly 415</td><td> Leu</td>

<td> Thr</td><td> Val</td><td> Phe</td><td> Glu 420</td><td> Asn</td><td> Tyr</td><td> Leu</td><td> Tyr</td><td> Ala 425</td><td> Thr</td><td> Asn</td><td> Ser</td><td> Asp</td><td> Asn 430</td><td> Ala</td><td> Asn</td>

<td> Ala</td><td> Gin</td><td> Gin 435</td><td> Lys</td><td> Thr</td><td> Ser</td><td> Val</td><td> Ile 440</td><td> Arg</td><td> Val</td><td> Asn</td><td> Arg</td><td> Phe 445</td><td> Asn</td><td> Ser</td><td> Thr</td>

<td> Glu</td><td> Tyr 450</td><td> Gin</td><td> Val</td><td> Val</td><td> Thr</td><td> Arg 455</td><td> Val</td><td> Asp</td><td> Lys</td><td> Gly</td><td> Gly 460</td><td> Ala</td><td> Leu</td><td> His</td><td> Ile</td>

<td> Tyr 465</td><td> His</td><td> Gin</td><td> Arg</td><td> Arg</td><td> Gin 470</td><td> Pro</td><td> Arg</td><td> Val</td><td> Arg</td><td> Ser 475</td><td> His</td><td> Ala</td><td> Cys</td><td> Glu</td><td> Asn 480</td>

<td> Asp</td><td> Gin</td><td> Tyr</td><td> Gly</td><td> Lys 485</td><td> Pro</td><td> Gly</td><td> Gly</td><td> Cys</td><td> Ser 490</td><td> Asp</td><td> Ile</td><td> Cys</td><td> Leu</td><td> Leu 495</td><td> Ala</td>

~*V0 94/18227

PCT/DK94/00054

Asn Ser His Lys Ala Arg Thr 500

Gly Ser Asp Gly Lys Ser Cys 515

Val Tyr Gly Lys Gly Arg Pro 530 535

Ala Lys Val Pro Asp Glu His 545 550

Pro Arg Ala Leu Asp Phe His 565

Asp Thr Thr Ser Tyr Leu lie 580

Arg Glu Thr lie Leu Lys Asp 595

Val Asp Trp Met Gly Asp Asn 610 615

Lys Thr lie Ser Val Ala Arg 625 630

Thr Leu lie Glu Gly Lys Met 645

Pro Leu Asn Gly Trp Met Tyr 660

Asp Ser Arg Arg Gly Arg Leu 675

Arg Asp lie Phe Val Thr Ser 690 695

Ser Leu Asp lie Pro Ala Gly 705 710

Asp Arg lie Glu Thr lie Leu 725

Tyr Glu Gly Pro Glu Leu Asn 740

Asn Tyr Leu Phe Trp Thr Glu 755

Glu Arg Gly Val Gly Gly Ala 770 775

Glu Arg Pro Pro lie Phe Glu 785 790

133

Cys Arg Cys Arg Ser Gly Phe Ser Leu 505 510

Lys Lys Pro Glu His Glu Leu Phe Leu 520 525

Gly lie lie Arg Gly Met Asp Met Gly 540

Met lie Pro lie Glu Asn Leu Met Asn 555 560

Ala Glu Thr Gly Phe lie Tyr Phe Ala 570 575

Gly Arg Gin Lys lie Asp Gly Thr Glu 585 590

Gly lie His Asn Val Glu Gly Val Ala 600 605

Leu Tyr Trp Thr Asp Asp Gly Pro Lys 620

Leu Glu Lys Ala Ala Gin Thr Arg Lys 635 640

Thr His Pro Arg Ala lie Val Val Asp 650 655

Trp Thr Asp Trp Glu Glu Asp Pro Lys 665 670

Glu Arg Ala Trp Met Asp Gly Ser His 680 685

Lys Thr Val Leu Trp Pro Asn Gly Leu 700

Arg Leu Tyr Trp Val Asp Ala Phe Tyr 715 720

Leu Asn Gly Thr Asp Arg Lys lie Val 730 735

His Ala Phe Gly Leu Cys His His Gly 745 750

Tyr Arg Ser Gly Ser Val Tyr Arg Leu 760 765

Pro Pro Thr Val Thr Leu Leu Arg Ser 780 lie Arg Met Tyr Asp Ala Gin Gin Gin 795 800

WO 94/18227

PCT/DK94/00054

<td> Gin Val Gly Thr Asn 805</td><td> Lys</td><td> Cys Arg Val Asn Asn Gly Gly 810</td><td> Cys</td><td> Ser 815</td><td> Ser</td>

<td> Leu Cys Leu Ala Thr</td><td> Pro</td><td> Gly Ser Arg Gin Cys Ala Cys</td><td> Ala</td><td> Glu</td><td> Asp</td>

<td> 820</td><td></td><td> 825</td><td> 830</td><td></td><td></td>

<td> Gin Val Leu Asp Ala</td><td> Asp</td><td> Gly Val Thr Cys Leu Ala Asn</td><td> Pro</td><td> Ser</td><td> Tyr</td>

<td> 835</td><td></td><td> 840 845</td><td></td><td></td><td></td>

<td> Val Pro Pro Pro Gin</td><td> Cys</td><td> Gin Pro Gly Glu Phe Ala Cys</td><td> Ala</td><td> Asn</td><td> Ser</td>

<td> 850</td><td></td><td> 855 860</td><td></td><td></td><td></td>

<td> Arg Cys Ile Gin Glu</td><td> Arg</td><td> Trp Lys Cys Asp Gly Asp Asn</td><td> Asp</td><td> Cys</td><td> Leu</td>

<td> 865</td><td> 870</td><td> 875</td><td></td><td></td><td> 880</td>

<td> Asp Asn Ser Asp Glu</td><td> Ala</td><td> Pro Ala Leu Cys His Gin His</td><td> Thr</td><td> Cys</td><td> Pro</td>

<td> 885</td><td></td><td> 890</td><td></td><td> 895</td><td></td>

<td> Ser Asp Arg Phe Lys</td><td> Cys</td><td> Glu Asn Asn Arg Cys He Pro</td><td> Asn</td><td> Arg</td><td> Trp</td>

<td> 900</td><td></td><td> 905</td><td> 910</td><td></td><td></td>

<td> Leu Cys Asp Gly Asp</td><td> Asn</td><td> Asp Cys Gly Asn Ser Glu Asp</td><td> Glu</td><td> Ser</td><td> Asn</td>

<td> 915</td><td></td><td> 920 925</td><td></td><td></td><td></td>

<td> Ala Thr Cys Ser Ala</td><td> Arg</td><td> Thr Cys Pro Pro Asn Gin Phe</td><td> Ser</td><td> Cys</td><td> Ala</td>

<td> 930</td><td></td><td> 935 940</td><td></td><td></td><td></td>

<td> Ser Gly Arg Cys He</td><td> Pro</td><td> lie Ser Trp Thr Cys Asp Leu</td><td> Asp</td><td> Asp</td><td> Asp</td>

<td> 945</td><td> 950</td><td> 955</td><td></td><td></td><td> 960</td>

<td> Cys Gly Asp Arg Ser</td><td> Asp</td><td> Glu Ser Ala Ser Cys Ala Tyr</td><td> Pro</td><td> Thr</td><td> Cys</td>

<td> 965</td><td></td><td> 970</td><td></td><td> 975</td><td></td>

<td> Phe Pro Leu Thr Gin</td><td> Phe</td><td> Thr Cys Asn Asn Gly Arg Cys</td><td> He</td><td> Asn</td><td> He</td>

<td> 980</td><td></td><td> 985</td><td> 990</td><td></td><td></td>

<td> Asn Trp Arg Cys Asp</td><td> Asn</td><td> Asp Asn Asp Cys Gly Asp Asn</td><td> Ser</td><td> Asp</td><td> Glu</td>

<td> 995</td><td></td><td colspan="2"> 1000 1005</td><td></td><td></td>

<td> Ala Gly Cys Ser His</td><td> Ser</td><td> Cys Ser Ser Thr Gin Phe Lys</td><td> Cys</td><td> Asn</td><td> Ser</td>

<td> 1010</td><td></td><td> 1015 1020</td><td></td><td></td><td></td>

<td> Gly Arg Cys lie Pro</td><td> Glu</td><td> His Trp Thr Cys Asp Gly Asp</td><td> Asn</td><td> Asp</td><td> Cys</td>

<td> 1025</td><td colspan="2"> 1030 1035</td><td></td><td></td><td> 1040</td>

<td> Gly Asp Tyr Ser Asp</td><td> Glu</td><td> Thr His Ala Asn Cys Thr Asn</td><td> Gin</td><td> Ala</td><td> Thr</td>

<td colspan="2"> 1045</td><td> 1050</td><td></td><td colspan="2"> 1055</td>

<td> Arg Pro Pro Gly Gly</td><td> Cys</td><td> His Thr Asp Glu Phe Gin Cys</td><td> Arg</td><td> Leu</td><td> Asp</td>

<td> 1060</td><td></td><td> 1065</td><td colspan="2"> 1070</td><td></td>

<td> Gly Leu Cys lie Pro</td><td> Leu</td><td> Arg Trp Arg Cys Asp Gly Asp</td><td> Thr</td><td> Asp</td><td> Cys</td>

<td> 1075</td><td></td><td colspan="2"> 1080 1085</td><td></td><td></td>

Met Asp Ser Ser Asp Glu Lys Ser Cys Glu Gly Val Thr His Val Cys 1090 1095 1100

-*V0 94/18227

PCT/DK94/00054

135

<td rowspan="2"> Asp Pro 1105</td><td rowspan="2"> Ser Val</td><td rowspan="2"> Lys</td><td colspan="2"> Phe Gly Cys Lys Asp Ser Ala Arg Cys</td><td rowspan="2"> He</td><td rowspan="2"> Ser 1120</td>

<td> 1110</td><td> 1115</td>

<td> Lys Ala</td><td> Trp Val</td><td> Cys</td><td> Asp Gly</td><td> Asp Asn Asp Cys Glu Asp Asn</td><td> Ser</td><td> Asp</td>

<td></td><td></td><td colspan="2"> 1125</td><td> 1130</td><td colspan="2"> 1135</td>

<td> Glu Glu</td><td> Asn Cys</td><td> Glu</td><td> Ser Leu</td><td> Ala Cys Arg Pro Pro Ser His</td><td> Pro</td><td> Cys</td>

<td></td><td colspan="2"> 1140</td><td></td><td colspan="2"> 1145 1150</td><td></td>

<td> Ala Asn</td><td> Asn Thr</td><td> Ser</td><td> Val Cys</td><td> Leu Pro Pro Asp Lys Leu Cys</td><td> Asp</td><td> Gly</td>

<td></td><td> 1155</td><td></td><td></td><td> 1160 1165</td><td></td><td></td>

<td> Asn Asp</td><td> Asp Cys</td><td> Gly</td><td> Asp Gly</td><td> Ser Asp Glu Gly Glu Leu Cys</td><td> Asp</td><td> Gin</td>

<td colspan="2"> 1170</td><td></td><td colspan="2"> 1175 1180</td><td></td><td></td>

<td> Cys Ser</td><td> Leu Asn</td><td> Asn</td><td> Gly Gly</td><td> Cys Ser His Asn Cys Ser Val</td><td> Ala</td><td> Pro</td>

<td> 1185</td><td></td><td></td><td> 1190</td><td> 1195</td><td></td><td> 1200</td>

<td> Gly Glu</td><td> Gly He</td><td> Val</td><td> Cys Ser</td><td> Cys Pro Leu Gly Met Glu Leu</td><td> Gly</td><td> Pro</td>

<td></td><td></td><td colspan="2"> 1205</td><td> 1210</td><td colspan="2"> 1215</td>

<td> Asp Asn</td><td> His Thr</td><td> Cys</td><td> Gin He</td><td> Gin Ser Tyr Cys Ala Lys His</td><td> Leu</td><td> Lys</td>

<td></td><td colspan="2"> 1220</td><td></td><td colspan="2"> 1225 1230</td><td></td>

<td> Cys Ser</td><td> Gin Lys</td><td> Cys</td><td> Asp Gin</td><td> Asn Lys Phe Ser Val Lys Cys</td><td> Ser</td><td> Cys</td>

<td></td><td> 1235</td><td></td><td></td><td> 1240 1245</td><td></td><td></td>

<td> Tyr Glu</td><td> Gly Trp</td><td> Val</td><td> Leu Glu</td><td> Pro Asp Gly Glu Ser Cys Arg</td><td> Ser</td><td> Leu</td>

<td colspan="2"> 1250</td><td></td><td> 125 =</td><td> &gt; 1260</td><td></td><td></td>

<td> Asp Pro</td><td> Phe Lys</td><td> Pro</td><td> Phe He</td><td> lie Phe Ser Asn Arg His Glu</td><td> He</td><td> Arg</td>

<td> 1265</td><td></td><td></td><td> 1270</td><td> 1275</td><td></td><td> 1280</td>

<td> Arg lie</td><td> Asp Leu</td><td> His</td><td> Lys Gly</td><td> Asp Tyr Ser Val Leu Val Pro</td><td> Gly</td><td> Leu</td>

<td></td><td></td><td colspan="2"> 1285</td><td> 1290</td><td colspan="2"> 1295</td>

<td> Arg Asn</td><td> Thr He</td><td> Ala</td><td> Leu Asp</td><td> Phe His Leu Ser Gin Ser Ala</td><td> Leu</td><td> Tyr</td>

<td></td><td colspan="2"> 1300</td><td></td><td colspan="2"> 1305 1310</td><td></td>

<td> Trp Thr</td><td> Asp Val</td><td> Val</td><td> Glu Asp</td><td> Lys He Tyr Arg Gly Lys Leu</td><td> Leu</td><td> Asp</td>

<td></td><td> 1315</td><td></td><td></td><td> 1320 1325</td><td></td><td></td>

<td> Asn Gly</td><td> Ala Leu</td><td> Thr</td><td> Ser Phe</td><td> Glu Val Val He Gin Tyr Gly</td><td> Leu</td><td> Ala</td>

<td colspan="2"> 1330</td><td></td><td> 133 =</td><td> ; 1340</td><td></td><td></td>

<td> Thr Pro</td><td> Glu Gly</td><td> Leu</td><td> Ala Val</td><td> Asp Trp He Ala Gly Asn He</td><td> Tyr</td><td> Trp</td>

<td> 1345 '</td><td></td><td></td><td> 1350</td><td> 1355</td><td></td><td> 1360</td>

<td> Val Glu</td><td> Ser Asn</td><td> Leu</td><td> Asp Gin</td><td> He Glu Val Ala Lys Leu Asp</td><td> Gly</td><td> Thr</td>

<td></td><td></td><td colspan="2"> 1365</td><td> 1370</td><td colspan="2"> 1375</td>

<td> Leu Arg</td><td> Thr Thr</td><td> Leu</td><td> Leu Ala</td><td> Gly Asp He Glu His Pro Arg</td><td> Ala</td><td> He</td>

1380 1385 1390

Ala Leu Asp Pro Arg Asp Gly lie Leu Phe Trp Thr Asp Trp Asp Ala 1395 1400 1405

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<img file="CA2155335C_D0026.tif" />

<td> Ser</td><td> Leu Pro Arg 1410</td><td> Ile</td><td> Glu</td><td> Ala Ala Ser Met 1415</td><td> Ser</td><td> Gly Ala Gly 1420</td><td> Arg</td><td> Arg</td>

<td> Thr</td><td> Val His Arg</td><td> Glu</td><td> Thr</td><td> Gly Ser Gly Gly</td><td> Trp</td><td> Pro Asn Gly</td><td> Leu</td><td> Thr</td>

<td colspan="2"> 1425</td><td></td><td colspan="2"> 1430</td><td colspan="2"> 1435</td><td></td><td> 1440</td>

<td> Val</td><td> Asp Tyr Leu</td><td> Glu</td><td> Lys</td><td> Arg Ile Leu Trp</td><td> Ile</td><td> Asp Ala Arg</td><td> Ser</td><td> Asp</td>

<td></td><td></td><td colspan="2"> 1445</td><td colspan="2"> 1450</td><td></td><td colspan="2"> 1455</td>

<td> Al a</td><td> Ile Tyr Ser</td><td> Ala</td><td> Arg</td><td> Tyr Asp Gly Ser</td><td> Gly</td><td> His Met Glu</td><td> Val</td><td> Leu</td>

<td></td><td colspan="2"> 1460</td><td></td><td> 1465</td><td></td><td colspan="2"> 1470</td><td></td>

<td> Arg</td><td> Gly His Glu</td><td> Phe</td><td> Leu</td><td> Ser His Pro Phe</td><td> Ala</td><td> Val Thr Leu</td><td> Tyr</td><td> Gly</td>

<td></td><td> 1475</td><td></td><td></td><td> 1480</td><td></td><td> 1485</td><td></td><td></td>

<td> Gly</td><td> Glu Val Tyr</td><td> Trp</td><td> Thr</td><td> Asp Trp Arg Thr</td><td> Asn</td><td> Thr Leu Ala</td><td> Lys</td><td> Ala</td>

<td></td><td> 1490</td><td></td><td></td><td> 1495</td><td></td><td> 1500</td><td></td><td></td>

<td> Asn</td><td> Lys Trp Thr</td><td> Gly</td><td> His</td><td> Asn Val Thr Val</td><td> Val</td><td> Gin Arg Thr</td><td> Asn</td><td> Thr</td>

<td colspan="2"> 1505</td><td></td><td colspan="2"> 1510</td><td colspan="2"> 1515</td><td></td><td> 1520</td>

<td> Gin</td><td> Pro Phe Asp</td><td> Leu</td><td> Gin</td><td> Val Tyr His Pro</td><td> Ser</td><td> Arg Gin Pro</td><td> Met</td><td> Ala</td>

<td></td><td></td><td colspan="2"> 1525</td><td colspan="2"> 1530</td><td></td><td colspan="2"> 1535</td>

<td> Pro</td><td> Asn Pro Cys</td><td> Glu</td><td> Ala</td><td> Asn Gly Gly Gin</td><td> Gly</td><td> Pro Cys Ser</td><td> His</td><td> Leu</td>

<td></td><td colspan="2"> 1540</td><td></td><td> 1545</td><td></td><td colspan="2"> 1550</td><td></td>

<td> Cys</td><td> Leu lie Asn</td><td> Tyr</td><td> Asn</td><td> Arg Thr Val Ser</td><td> Cys</td><td> Ala Cys Pro</td><td> His</td><td> Leu</td>

<td></td><td> 1555</td><td></td><td></td><td> 1560</td><td></td><td> 1565</td><td></td><td></td>

<td> Met</td><td> Lys Leu His</td><td> Lys</td><td> Asp</td><td> Asn Thr Thr Cys</td><td> Tyr</td><td> Glu Phe Lys</td><td> Lys</td><td> Phe</td>

<td></td><td> 1570</td><td></td><td></td><td> 1575</td><td></td><td> 1580</td><td></td><td></td>

<td> Leu</td><td> Leu Tyr Ala</td><td> Arg</td><td> Gin</td><td> Met Glu Ile Arg</td><td> Gly</td><td> Val Asp Leu</td><td> Asp</td><td> Ala</td>

<td colspan="2"> 1585</td><td></td><td colspan="2"> 1590</td><td colspan="2"> 1595</td><td></td><td> 1600</td>

<td> Pro</td><td> Tyr Tyr Asn</td><td> Tyr</td><td> Ile</td><td> Ile Ser Phe Thr</td><td> Val</td><td> Pro Asp Ile</td><td> Asp</td><td> Asn</td>

<td></td><td></td><td colspan="2"> 1605</td><td colspan="2"> 1610</td><td></td><td colspan="2"> 1615</td>

<td> Val</td><td> Thr Val Leu</td><td> Asp</td><td> Tyr</td><td> Asp Ala Arg Glu</td><td> Gin</td><td> Arg Val Tyr</td><td> Trp</td><td> Ser</td>

<td></td><td colspan="2"> 1620</td><td></td><td> 1625</td><td></td><td colspan="2"> 1630</td><td></td>

<td> Asp</td><td> Val Arg Thr</td><td> Gin</td><td> Ala</td><td> Ile Lys Arg Ala</td><td> Phe</td><td> Ile Asn Gly</td><td> Thr</td><td> Gly</td>

<td></td><td> 1635</td><td></td><td></td><td> 1640</td><td></td><td> 1645</td><td></td><td></td>

<td> Val</td><td> Glu Thr Val</td><td> Val</td><td> Ser</td><td> Ala Asp Leu Pro</td><td> Asn</td><td> Ala His Gly</td><td> Leu</td><td> Ala</td>

<td></td><td> 1650</td><td></td><td></td><td> 1655</td><td></td><td> 1660</td><td></td><td></td>

<td> Val</td><td> Asp Trp Val</td><td> Ser</td><td> Arg</td><td> Asn Leu Phe Trp</td><td> Thr</td><td> Ser Tyr Asp</td><td> Thr</td><td> Asn</td>

<td colspan="2"> 1665</td><td></td><td colspan="2"> 1670</td><td colspan="2"> 1675</td><td></td><td> 1680</td>

<td> Lys</td><td> Lys Gin Ile</td><td> Asn</td><td> Val</td><td> Ala Arg Leu Asp</td><td> Gly</td><td> Ser Phe Lys</td><td> Asn</td><td> Ala</td>

1685 1690 1695

Val Val Gin Gly Leu Glu Gin Pro His Gly Leu Val Val His Pro Leu 1700 1705 1710 “*V0 94/18227

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<td colspan="4"> Arg Gly Lys Leu Tyr Trp Thr Asp Gly Asp Asn Ile Ser Met Ala Asn</td>

<td></td><td> 1715</td><td> 1720</td><td> 1725</td>

<td> Met Asp</td><td> Gly Ser Asn Arg Thr</td><td> Leu Leu</td><td> Phe Ser Gly Gin Lys Gly Pro</td>

<td colspan="3"> 1730 1735</td><td> 174Π</td>

<td> Val Gly</td><td> Leu Ala Ile Asp Phe</td><td> Pro Glu</td><td> Ser Lys Leu Tyr Trp Ile Ser</td>

<td> 1745</td><td> 1750</td><td></td><td> 1755 1760</td>

<td> Ser Gly</td><td> Asn His Thr Ile Asn</td><td> Arg Cys</td><td> Asn Leu Asp Gly Ser Gly Leu</td>

<td></td><td> 1765</td><td></td><td> 1770 1775</td>

<td> Glu Val</td><td> Ile Asp Ala Met Arg</td><td> Ser Gin</td><td> Leu Gly Lys Ala Thr Ala Leu</td>

<td></td><td> 1780</td><td colspan="2"> 1785 1790</td>

<td> Ala Ile</td><td> Met Gly Asp Lys Leu</td><td> Trp Trp</td><td> Ala Asp Gin Val Ser Glu Lys</td>

<td></td><td> 1795</td><td> 1800</td><td> 1805</td>

<td> Met Gly</td><td> Thr Cys Ser Lys Ala</td><td> Asp Gly</td><td> Ser Gly Ser Val Val Leu Arg</td>

<td colspan="3"> 1810 1815</td><td> 1820</td>

<td> Asn Ser</td><td> Thr Thr Leu Val Met</td><td> His Met</td><td> Lys Val Tyr Asp Glu Ser Ile</td>

<td> 1825</td><td> 1830</td><td></td><td> 1835 1840</td>

<td> Gin Leu</td><td> Asp His Lys Gly Thr</td><td> Asn Pro</td><td> Cys Ser Val Asn Asn Gly Asp</td>

<td></td><td> 1845</td><td></td><td> 1850 1855</td>

<td> Cys Ser</td><td> Gin Leu Cys Leu Pro</td><td> Thr Ser</td><td> Glu Thr Thr Arg Ser Cys Met</td>

<td></td><td> 1860</td><td colspan="2"> 1865 1870</td>

<td> Cys Thr</td><td> Ala Gly Tyr Ser Leu</td><td> Arg Ser</td><td> Gly Gin Gin Ala Cys Glu Gly</td>

<td></td><td> 1875</td><td> 1880</td><td> 1885</td>

<td> Val Gly</td><td> Ser Phe Leu Leu Tyr</td><td> Ser Val</td><td> His Glu Gly Ile Arg Gly Ile</td>

<td colspan="3"> 1890 1895</td><td> 1900</td>

<td> Pro Leu</td><td> Asp Pro Asn Asp Lys</td><td> Ser Asp</td><td> Ala Leu Val Pro Val Ser Gly</td>

<td> 1905</td><td> 1910</td><td></td><td> 1915 1920</td>

<td> Thr Ser</td><td> Leu Ala Val Gly Ile</td><td> Asp Phe</td><td> His Ala Glu Asn Asp Thr Ile</td>

<td></td><td> 1925</td><td></td><td> 1930 1935</td>

<td> Tyr Trp</td><td> Val Asp Met Gly Leu</td><td> Ser Thr</td><td> Ile Ser Arg Ala Lys Arg Asp</td>

<td></td><td> 1940</td><td colspan="2"> 1945 1950</td>

<td> Gin Thr</td><td> Trp Arg Glu Asp Val</td><td> Val Thr</td><td> Asn Gly Ile Gly Arg Val Glu</td>

<td></td><td> 1955</td><td> 1960</td><td> 1965</td>

<td> Gly Ile</td><td> Ala Val Asp Trp Ile</td><td> Ala Gly</td><td> Asn Ile Tyr Trp Thr Asp Gin</td>

<td colspan="3"> 1970 1975</td><td> 1980</td>

<td> Gly Phe</td><td> Asp Val Ile Glu Val</td><td> Ala Arg</td><td> Leu Asn Gly Ser Phe Arg Tyr</td>

1985

1990

1995

2000

Val Val Ile Ser Gin Gly Leu Asp Lys Pro Arg Ala Ile Thr Val His 2005 2010 2015

<img file="CA2155335C_D0027.tif" />

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<td> Pro</td><td> Glu Lys Gly Tyr 2020</td><td> Leu</td><td> Phe Trp Thr Glu 2025</td><td> Trp</td><td> Gly Gin Tyr Pro 2030</td><td> Arg</td>

<td> Ile</td><td> Glu Arg Ser Arg 2035</td><td> Leu</td><td> Asp Gly Thr Glu 2040</td><td> Arg</td><td> Val Val Leu Val 2045</td><td> Asn</td>

<td> Val</td><td> Ser Ile Ser Trp 2050</td><td> Pro</td><td> Asn Gly Ile Ser 2055</td><td> Val</td><td> Asp Tyr Gin Asp 2060</td><td> Gly</td>

<td> Lys</td><td> Leu Tyr Trp Cys</td><td> Asp</td><td> Ala Arg Thr Asp</td><td> Lys</td><td> Ile Glu Arg Ile</td><td> Asp</td>

<td colspan="2"> 2065</td><td colspan="2"> 2070</td><td colspan="2"> 2075</td><td> 2080</td>

<td> Leu</td><td> Glu Thr Gly Glu</td><td> Asn</td><td> Arg Glu Val Val</td><td> Leu</td><td> Ser Ser Asn Asn</td><td> Met</td>

<td></td><td colspan="2"> 2085</td><td colspan="2"> 2090</td><td colspan="2"> 2095</td>

<td> Asp</td><td> Met Phe Ser Val 2100</td><td> Ser</td><td> Val Phe Glu Asp 2105</td><td> Phe</td><td> Ile Tyr Trp Ser 2110</td><td> Asp</td>

<td> Arg</td><td> Thr His Ala Asn 2115</td><td> Gly</td><td> Ser Ile Lys Arg 2120</td><td> Gly</td><td> Ser Lys Asp Asn 2125</td><td> Ala</td>

<td> Thr</td><td> Asp Ser Val Pro 2130</td><td> Leu</td><td> Arg Thr Gly Ile 2135</td><td> Gly</td><td> Val Gin Leu Lys 2140</td><td> Asp</td>

<td> Ile</td><td> Lys Val Phe Asn</td><td> Arg</td><td> Asp Arg Gin Lys</td><td> Gly</td><td> Thr Asn Val Cys</td><td> Ala</td>

<td colspan="2"> 2145</td><td colspan="2"> 2150</td><td colspan="2"> 2155</td><td> 2160</td>

<td> Val</td><td> Ala Asn Gly Gly</td><td> Cys</td><td> Gin Gin Leu Cys</td><td> Leu</td><td> Tyr Arg Gly Arg</td><td> Gly</td>

<td></td><td colspan="2"> 2165</td><td colspan="2"> 2170</td><td colspan="2"> 2175</td>

<td> Gin</td><td> Arg Ala Cys Ala 2180</td><td> Cys</td><td> Ala His Gly Met 2185</td><td> Leu</td><td> Ala Glu Asp Gly 2190</td><td> Ala</td>

<td> Ser</td><td> Cys Arg Glu Tyr 2195</td><td> Ala</td><td> Gly Tyr Leu Leu 2200</td><td> Tyr</td><td> Ser Glu Arg Thr 2205</td><td> Ile</td>

<td> Leu</td><td> Lys Ser Ile His 2210</td><td> Leu</td><td> Ser Asp Glu Arg 2215</td><td> Asn</td><td> Leu Asn Ala Pro 2220</td><td> Val</td>

<td> Gin</td><td> Pro Phe Glu Asp</td><td> Pro</td><td> Glu His Met Lys</td><td> Asn</td><td> Val Ile Ala Leu</td><td> Ala</td>

<td colspan="2"> 2225</td><td colspan="2"> 2230</td><td colspan="2"> 2235</td><td> 2240</td>

<td> Phe</td><td> Asp Tyr Arg Ala</td><td> Gly</td><td> Thr Ser Pro Gly</td><td> Thr</td><td> Pro Asn Arg Ile</td><td> Phe</td>

<td></td><td colspan="2"> 2245</td><td colspan="2"> 2250</td><td colspan="2"> 2255</td>

<td> Phe</td><td> Ser Asp Ile His 2260</td><td> Phe</td><td> Gly Asn Ile Gin 2265</td><td> Gin</td><td> Ile Asn Asp Asp 2270</td><td> Gly</td>

<td> Ser</td><td> Arg Arg Ile Thr 2275</td><td> Ile</td><td> Val Glu Asn Val 2280</td><td> Gly</td><td> Ser Val Glu Gly 2285</td><td> Leu</td>

<td> Al a</td><td> Tyr His Arg Gly 2290</td><td> Trp</td><td> Asp Thr Leu Tyr 2295</td><td> Trp</td><td> Thr Ser Tyr Thr 2300</td><td> Thr</td>

<td> Ser</td><td> Thr Ile Thr Arg</td><td> His</td><td> Thr Val Asp Gin</td><td> Thr</td><td> Arg Pro Gly Ala</td><td> Phe</td>

<td colspan="2"> 2305</td><td colspan="2"> 2310</td><td colspan="2"> 2315</td><td> 2320</td>

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<td> Glu</td><td> Arg</td><td> Glu</td><td> Thr</td><td> Val Ile Thr Met 2325</td><td> Ser</td><td> Gly Asp 2330</td><td> Asp</td><td> His</td><td> Pro Arg Ala 2335</td>

<td> Phe</td><td> Val</td><td> Leu</td><td> Asp</td><td> Glu Cys Gin Asn</td><td> Leu</td><td> Met Phe</td><td> Trp</td><td> Thr</td><td> Asn Trp Asn</td>

<td></td><td></td><td></td><td colspan="2"> 2340</td><td colspan="2"> 2345</td><td></td><td></td><td> 2350</td>

<td> «.ilU</td><td> Gin</td><td> His</td><td> Pro</td><td> Ser Ile Met Arg</td><td> Ala</td><td> Ala Leu</td><td> Ser</td><td> Gly</td><td> Ala Asn Val</td>

<td></td><td></td><td colspan="2"> 2355</td><td colspan="2"> 2360</td><td></td><td></td><td colspan="2"> 2365</td>

<td> Leu</td><td> Thr</td><td> Leu</td><td> Ile</td><td> Glu Lys Asp Ile</td><td> Arg</td><td> Thr Pro</td><td> Asn</td><td> Gly</td><td> Leu Ala Ile</td>

<td></td><td colspan="2"> 2370</td><td></td><td> 2375</td><td></td><td></td><td colspan="2"> 2380</td><td></td>

<td> Asp</td><td> His</td><td> Arg</td><td> Ala</td><td> Glu Lys Leu Tyr</td><td> Phe</td><td> Ser Asp</td><td> Ala</td><td> Thr</td><td> Leu Asp Lys</td>

<td colspan="2"> 2385</td><td></td><td></td><td> 2390</td><td></td><td colspan="2"> 2395</td><td></td><td> 2400</td>

<td> Ile</td><td> Glu</td><td> Arg</td><td> Cys</td><td> Glu Tyr Asp Gly</td><td> Ser</td><td> His Arg</td><td> Tyr</td><td> Val</td><td> Ile Leu Lys</td>

<td></td><td></td><td></td><td></td><td> 2405</td><td></td><td> 2410</td><td></td><td></td><td> 2415</td>

<td> Ser</td><td> Glu</td><td> Pro</td><td> Val</td><td> His Pro Phe Gly</td><td> Leu</td><td> Ala Val</td><td> Tyr</td><td> Gly</td><td> Glu His Ile</td>

<td></td><td></td><td></td><td colspan="2"> 2420</td><td colspan="2"> 2425</td><td></td><td></td><td> 2430</td>

<td> Phe</td><td> Trp</td><td> Thr</td><td> Asp</td><td> Trp Val Arg Arg</td><td> Ala</td><td> Val Gin</td><td> Arg</td><td> Ala</td><td> Asn Lys His</td>

<td></td><td></td><td colspan="2"> 2435</td><td colspan="2"> 2440</td><td></td><td></td><td colspan="2"> 2445</td>

<td> Val</td><td> Gly</td><td> Ser</td><td> Asn</td><td> Met Lys Leu Leu</td><td> Arg</td><td> Val Asp</td><td> Ile</td><td> Pro</td><td> Gin Gin Pro</td>

<td></td><td colspan="2"> 2450</td><td></td><td> 2455</td><td></td><td></td><td colspan="2"> 2460</td><td></td>

<td> Met</td><td> Gly</td><td> Ile</td><td> Ile</td><td> Ala Val Ala Asn</td><td> Asp</td><td> Thr Asn</td><td> Ser</td><td> Cys</td><td> Glu Leu Ser</td>

<td colspan="2"> 2465</td><td></td><td></td><td> 2470</td><td></td><td colspan="2"> 2475</td><td></td><td> 2480</td>

<td> Pro</td><td> Cys</td><td> Arg</td><td> Ile</td><td> Asn Asn Gly Gly</td><td> Cys</td><td> Gin Asp</td><td> Leu</td><td> Cys</td><td> Leu Leu Thr</td>

<td></td><td></td><td></td><td></td><td> 2485</td><td></td><td> 2490</td><td></td><td></td><td> 2495</td>

<td> His</td><td> Gin</td><td> Gly</td><td> His</td><td> Val Asn Cys Ser</td><td> Cys</td><td> Arg Gly</td><td> Gly</td><td> Arg</td><td> Ile Leu Gin</td>

<td></td><td></td><td></td><td colspan="2"> 2500</td><td colspan="2"> 2505</td><td></td><td></td><td> 2510</td>

<td> Asp</td><td> Asp</td><td> Leu</td><td> Thr</td><td> Cys Arg Ala Val</td><td> Asn</td><td> Ser Ser</td><td> cys</td><td> Arg</td><td> Ala Gin Asp</td>

<td></td><td></td><td colspan="2"> 2515</td><td colspan="2"> 2520</td><td></td><td></td><td colspan="2"> 2525</td>

<td> Glu</td><td> Phe</td><td> Glu</td><td> Cys</td><td> Ala Asn Gly Glu</td><td> Cys</td><td> Ile Asn</td><td> Phe</td><td> Ser</td><td> Leu Thr Cys</td>

<td></td><td colspan="2"> 2530</td><td></td><td> 2535</td><td></td><td></td><td colspan="2"> 2540</td><td></td>

<td> Asp</td><td> Gly</td><td> Val</td><td> Pro</td><td> His Cys Lys Asp</td><td> Lys</td><td> Ser Asp</td><td> Glu</td><td> Lys</td><td> Pro Ser Tyr</td>

<td colspan="2"> 2545</td><td></td><td></td><td> 2550</td><td></td><td colspan="2"> 2555</td><td></td><td> 2560</td>

<td> Cys</td><td> Asn</td><td> Ser</td><td> Arg</td><td> Arg Cys Lys Lys</td><td> Thr</td><td> Phe Arg</td><td> Gin</td><td> Cys</td><td> Ser Asn Gly</td>

<td></td><td></td><td></td><td></td><td> 2565</td><td></td><td> 2570</td><td></td><td></td><td> 2575</td>

<td> Arg</td><td> Cys</td><td> Val</td><td> Ser</td><td> Asn Met Leu Trp</td><td> Cys</td><td> Asn Gly</td><td> Ala</td><td> Asp</td><td> Asp Cys Gly</td>

<td></td><td></td><td></td><td colspan="2"> 2580</td><td colspan="2"> 2585</td><td></td><td></td><td> 2590</td>

<td> Asp</td><td> Gly</td><td> Ser</td><td> Asp</td><td> Glu Ile Pro Cys</td><td> Asn</td><td> Lys Thr</td><td> Ala</td><td> Cys</td><td> Gly Val Gly</td>

2595 2600 2605

Glu Phe Arg Cys Arg Asp Gly Thr Cys Ile Gly Asn Ser Ser Arg Cys 2610 2615 2620

<img file="CA2155335C_D0028.tif" />

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<td rowspan="2"> Asn Gin Phe 2625</td><td rowspan="2"> Val Asp</td><td colspan="2"> Cys Glu Asp Ala Ser Asp Glu Met Asn Cys</td><td rowspan="2"> Ser 2640</td>

<td> 2630</td><td> 2635</td>

<td> Ala Thr Asp</td><td> Cys Ser</td><td> Ser Tyr Phe Arg</td><td> Leu Gly Val Lys Gly Val</td><td> Leu</td>

<td></td><td colspan="2"> 2645</td><td colspan="2"> 2650 2655</td>

<td> Phe Gin Pro</td><td> Cys Glu</td><td> Arg Thr Ser Leu</td><td> Cys Tyr Ala t-ro Ser Trp</td><td> Val</td>

<td></td><td> 2660</td><td colspan="2"> 2665 2670</td><td></td>

<td> Cys Asp Gly</td><td> Ala Asn</td><td> Asp Cys Gly Asp</td><td> Tyr Ser Asp Glu Arg Asp</td><td> Cys</td>

<td colspan="2"> 2675</td><td> 2680</td><td> 2685</td><td></td>

<td> Pro Gly Val</td><td> Lys Arg</td><td> Pro Arg Cys Pro</td><td> Leu Asn Tyr Phe Ala Cys</td><td> Pro</td>

<td> 2690</td><td></td><td> 2695</td><td> 2700</td><td></td>

<td> Ser Gly Arg</td><td> Cys lie</td><td> Pro Met Ser Trp</td><td> Thr Cys Asp Lys Glu Asp</td><td> Asp</td>

<td> 2705</td><td></td><td> 2710</td><td> 2715</td><td> 2720</td>

<td> Cys Glu His</td><td> Gly Glu</td><td> Asp Glu Thr His</td><td> Cys Asn Lys Phe Cys Ser</td><td> Glu</td>

<td></td><td colspan="2"> 2725</td><td colspan="2"> 2730 2735</td>

<td> Ala Gin Phe</td><td> Glu Cys</td><td> Gin Asn His Arg</td><td> Cys He Ser Lys Gin Trp</td><td> Leu</td>

<td></td><td> 2740</td><td colspan="2"> 2745 2750</td><td></td>

<td> Cys Asp Gly</td><td> Ser Asp</td><td> Asp Cys Gly Asp</td><td> Gly Ser Asp Glu Ala Ala</td><td> His</td>

<td colspan="2"> 2755</td><td> 2760</td><td> 2765</td><td></td>

<td> Cys Glu Gly</td><td> Lys Thr</td><td> Cys Gly Pro Ser</td><td> Ser Phe Ser Cys Pro Gly</td><td> Thr</td>

<td> 2770</td><td></td><td> 2775</td><td> 2780</td><td></td>

<td> His Val Cys</td><td> Val Pro</td><td> Glu Arg Trp Leu</td><td> Cys Asp Gly Asp Lys Asp</td><td> Cys</td>

<td> 2785</td><td></td><td> 2790</td><td> 2795</td><td> 2800</td>

<td> Ala Asp Gly</td><td> Ala Asp</td><td> Glu Ser He Ala</td><td> Ala Gly Cys Leu Tyr Asn</td><td> Ser</td>

<td></td><td colspan="2"> 2805</td><td colspan="2"> 2810 2815</td>

<td> Thr Cys Asp</td><td> Asp Arg</td><td> Glu Phe Met Cys</td><td> Gin Asn Arg Gin Cys He</td><td> Pro</td>

<td></td><td> 2820</td><td colspan="2"> 2825 2830</td><td></td>

<td> Lys His Phe</td><td> Val Cys</td><td> Asp His Asp Arg</td><td> Asp Cys Ala Asp Gly Ser</td><td> Asp</td>

<td colspan="2"> 2835</td><td> 2840</td><td> 2845</td><td></td>

<td> Glu Ser Pro</td><td> Glu Cys</td><td> Glu Tyr Pro Thr</td><td> Cys Gly Pro Ser Glu Phe</td><td> Arg</td>

<td> 2850</td><td></td><td> 2855</td><td> 2860</td><td></td>

<td> Cys Ala Asn</td><td> Gly Arg</td><td> Cys Leu Ser Ser</td><td> Arg Gin Trp Glu Cys Asp</td><td> Gly</td>

<td> 2865</td><td></td><td> 2670</td><td> 2875</td><td> 2880</td>

<td> Glu Asn Asp</td><td> Cys His</td><td> Asp Gin Ser Asp</td><td> Glu Ala Pro Lys Asn Pro</td><td> His</td>

<td></td><td colspan="2"> 2885</td><td colspan="2"> 2890 2895</td>

<td> Cys Thr Ser</td><td> Pro Glu</td><td> His Lys Cys Asn</td><td> Ala Ser Ser Gin Phe Leu</td><td> Cys</td>

2900 2905 2910

Ser Ser Gly Arg Cys Val Ala Glu Ala Leu Leu Cys Asn Gly Gin Asp 2915 2920 2925

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<td> Asp</td><td> Cys Gly 2930</td><td> Asp</td><td> Ser</td><td> Ser</td><td> Asp Glu 2935</td><td> Arg</td><td> Gly</td><td> Cys</td><td> His lie 2940</td><td> Asn</td><td> Glu</td><td> Cys</td>

<td> Leu</td><td> Ser Arg</td><td> Lys</td><td> Leu</td><td> Ser</td><td> Gly Cys</td><td> Ser</td><td> Gin</td><td> Asp</td><td> Cys Glu</td><td> Asp</td><td> Leu</td><td> Lys</td>

<td colspan="2"> 2945</td><td></td><td></td><td colspan="2"> 2950</td><td></td><td></td><td colspan="2"> 2955</td><td></td><td></td><td> 2960</td>

<td> lie</td><td> Gly Phe</td><td> Lys</td><td> Cys</td><td> Arg</td><td> C&gt;s Arg</td><td> Pro</td><td> Gly</td><td> Phe</td><td> Arg Leu</td><td> Lys</td><td> Asp</td><td> Asp</td>

<td></td><td></td><td></td><td colspan="2"> 2965</td><td></td><td></td><td colspan="2"> 2970</td><td></td><td></td><td colspan="2"> 2975</td>

<td> Gly</td><td> Arg Thr</td><td> cys</td><td> Ala</td><td> Asp</td><td> Val Asp</td><td> Glu</td><td> Cys</td><td> Ser</td><td> Thr Thr</td><td> Phe</td><td> Pro</td><td> Cys</td>

<td></td><td></td><td colspan="2"> 2980</td><td></td><td></td><td colspan="2"> 2985</td><td></td><td></td><td colspan="2"> 2990</td><td></td>

<td> Ser</td><td> Gin Arg</td><td> Cys</td><td> He</td><td> Asn</td><td> Thr His</td><td> Gly</td><td> Ser</td><td> Tyr</td><td> Lys Cys</td><td> Leu</td><td> Cys</td><td> Val</td>

<td></td><td colspan="2"> 2995</td><td></td><td></td><td colspan="2"> 3000</td><td></td><td></td><td colspan="2"> 3005</td><td></td><td></td>

<td> Glu</td><td> Gly Tyr</td><td> Ala</td><td> Pro</td><td> Arg</td><td> Gly Gly</td><td> Asp</td><td> Pro</td><td> His</td><td> Ser Cys</td><td></td><td> Ala</td><td> Val</td>

<td></td><td> 3010</td><td></td><td></td><td></td><td> 3015</td><td></td><td></td><td></td><td> 3020</td><td></td><td></td><td></td>

<td> Thr</td><td> Asp Glu</td><td> Glu</td><td> Pro</td><td> Phe</td><td> Leu He</td><td> Phe</td><td> Ala</td><td> Asn</td><td> Arg Tyr</td><td> Tyr</td><td> Leu</td><td> Arg</td>

<td colspan="2"> 3025</td><td></td><td></td><td colspan="2"> 3030</td><td></td><td></td><td colspan="2"> 3035</td><td></td><td></td><td> 3040</td>

<td> Lys</td><td> Leu Asn</td><td> Leu</td><td> Asp</td><td> Gly</td><td> Ser Asn</td><td> Tyr</td><td> Thr</td><td> Leu</td><td> Leu Lys</td><td> Gin</td><td> Gly</td><td> Leu</td>

<td></td><td></td><td></td><td colspan="2"> 3045</td><td></td><td></td><td colspan="2"> 3050</td><td></td><td></td><td colspan="2"> 3055</td>

<td> Asn</td><td> Asn Ala</td><td> Val</td><td> Ala</td><td> Leu</td><td> Asp Phe</td><td> Asp</td><td> Tyr</td><td> Arg</td><td> Glu Gin</td><td> Met</td><td> He</td><td> Tyr</td>

<td></td><td></td><td colspan="2"> 3060</td><td></td><td></td><td colspan="2"> 3065</td><td></td><td></td><td colspan="2"> 3070</td><td></td>

<td> Trp</td><td> Thr Asp</td><td> Val</td><td> Thr</td><td> Thr</td><td> Gin Gly</td><td> Ser</td><td> Met</td><td> He</td><td> Arg Arg</td><td> Met</td><td> His</td><td> Leu</td>

<td></td><td colspan="2"> 3075</td><td></td><td></td><td colspan="2"> 3080</td><td></td><td></td><td colspan="2"> 3085</td><td></td><td></td>

<td> Asn</td><td> Gly Ser</td><td> Asn</td><td> Val</td><td> Gin</td><td> Val Leu</td><td> His</td><td> Arg</td><td> Thr</td><td> Gly Leu</td><td> Ser</td><td> Asn</td><td> Pro</td>

<td></td><td> 3090</td><td></td><td></td><td></td><td> 3095</td><td></td><td></td><td></td><td> 3100</td><td></td><td></td><td></td>

<td> ASp</td><td> Gly Leu</td><td> Ala</td><td> Val</td><td> Asp</td><td> Trp Val</td><td> Gly</td><td> Gly</td><td> Asn</td><td> Leu Tyr</td><td> Trp</td><td> Cys</td><td> Asp</td>

<td colspan="2"> 3105</td><td></td><td></td><td colspan="2"> 3110</td><td></td><td></td><td colspan="2"> 3115</td><td></td><td></td><td> 3120</td>

<td> Lys</td><td> Gly Arg</td><td> Asp</td><td> Thr</td><td> He</td><td> Glu Val</td><td> Ser</td><td> Lys</td><td> Leu</td><td> Asn Gly</td><td> Ala</td><td> Tyr</td><td> Arg</td>

<td></td><td></td><td> •</td><td colspan="2"> 3125</td><td></td><td></td><td colspan="2"> 3130</td><td></td><td></td><td colspan="2"> 3135</td>

<td> Thr</td><td> Val Leu</td><td> Val</td><td> Ser</td><td> Ser</td><td> Gly Leu</td><td> Arg</td><td> Glu</td><td> Pro</td><td> Arg Ala</td><td> Leu</td><td> Val</td><td> Val</td>

<td></td><td></td><td colspan="2"> 3140</td><td></td><td></td><td colspan="2"> 3145</td><td></td><td></td><td colspan="2"> 3150</td><td></td>

<td> Asp</td><td> Val Gin</td><td> Asn</td><td> Gly</td><td> Tyr</td><td> Leu Tyr</td><td> Trp</td><td> Thr</td><td> Asp</td><td> Trp Gly</td><td> Asp</td><td> His</td><td> Ser</td>

<td></td><td colspan="2"> 3155</td><td></td><td></td><td colspan="2"> 3160</td><td></td><td></td><td colspan="2"> 3165</td><td></td><td></td>

<td> Leu</td><td> He Gly</td><td> Arg</td><td> He</td><td> Gly</td><td> Met Asp</td><td> Gly</td><td> Ser</td><td> Ser</td><td> Arg Ser</td><td> Val</td><td> He</td><td> Val</td>

<td></td><td> 3170</td><td></td><td></td><td></td><td> 3175</td><td></td><td></td><td></td><td> 3180</td><td></td><td></td><td></td>

<td> Asp</td><td> Thr Lys</td><td> He</td><td> Thr</td><td> Trp</td><td> Pro Asn</td><td> Gly</td><td> Leu</td><td> Thr</td><td> Leu Asp</td><td> Tyr</td><td> Val</td><td> Thr</td>

<td colspan="2"> 3185</td><td></td><td></td><td colspan="2"> 3190</td><td></td><td></td><td colspan="2"> 3195</td><td></td><td></td><td> 3200</td>

<td> Glu</td><td> Arg He</td><td> Tyr</td><td> Trp</td><td> Ala</td><td> Asp Ala</td><td> Arg</td><td> Glu</td><td> Asp</td><td> Tyr He</td><td> Glu</td><td> Phe</td><td> Ala</td>

3205 3210 3215

Ser Leu Asp Gly Ser Asn Arg His Val Val Leu Ser Gin Asp He Pro 3220 3225 3230

<img file="CA2155335C_D0029.tif" />

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<td> His Ile Phe Ala 3235</td><td> Leu</td><td> Thr Leu Phe Glu 3240</td><td> Asp</td><td> Tyr Val Tyr Trp 3245</td><td> Thr</td><td> Asp</td>

<td> Trp Glu Thr Lys 3250</td><td> Ser</td><td> lie Asn Arg Ala 3255</td><td> His</td><td> Lys Thr Thr Gly 3260</td><td> Thr</td><td> Asn</td>

<td> Lys Thr Leu Leu 3265</td><td> lie</td><td> Ser Thr Leu His 3270</td><td> Arg</td><td> Pro Met Asp Leu 3275</td><td> His</td><td> Val 3280</td>

<td> Phe His Ala Leu</td><td> Arg</td><td> Gin Pro Asp Val</td><td> Pro</td><td> Asn His Pro Cys</td><td> Lys</td><td> Val</td>

<td></td><td colspan="2"> 3285</td><td colspan="2"> 3290</td><td colspan="2"> 3295</td>

<td> Asn Asn Gly Gly</td><td> Cys</td><td> Ser Asn Leu Cys</td><td> Leu</td><td> Leu Ser Pro Gly</td><td> Gly</td><td> Gly</td>

<td colspan="2"> 3300</td><td colspan="2"> 3305</td><td colspan="2"> 3310</td><td></td>

<td> His Lys Cys Ala 3315</td><td> Cys</td><td> Pro Thr Asn Phe 3320</td><td> Tyr</td><td> Leu Gly Ser Asp 3325</td><td> Gly</td><td> Arg</td>

<td> Thr Cys Val Ser 3330</td><td> Asn</td><td> Cys Thr Ala Ser 3335</td><td> Gin</td><td> Phe Val Cys Lys 3340</td><td> Asn</td><td> Asp</td>

<td> Lys Cys lie Pro 3345</td><td> Phe</td><td> Trp Trp Lys Cys 3350</td><td> Asp</td><td> Thr Glu Asp Asp 3355</td><td> Cys</td><td> Gly 3360</td>

<td> Asp His Ser Asp</td><td> Glu</td><td> Pro Pro Asp Cys</td><td> Pro</td><td> Glu Phe Lys Cys</td><td> Arg</td><td> Pro</td>

<td></td><td colspan="2"> 3365</td><td colspan="2"> 3370</td><td colspan="2"> 3375</td>

<td> Gly Gin Phe Gin</td><td> Cys</td><td> Ser Thr Gly He</td><td> Cys</td><td> Thr Asn Pro Ala</td><td> Phe</td><td> He</td>

<td colspan="2"> 3380</td><td colspan="2"> 3385</td><td colspan="2"> 3390</td><td></td>

<td> Cys Asp Gly Asp 3395</td><td> Asn</td><td> Asp Cys Gin Asp 3400</td><td> Asn</td><td> Ser Asp Glu Ala 3405</td><td> Asn</td><td> Cys</td>

<td> Asp lie His Val 3410</td><td> Cys</td><td> Leu Pro Ser Gin 3415</td><td> Phe</td><td> Lys Cys Thr Asn 3420</td><td> Thr</td><td> Asn</td>

<td> Arg Cys lie Pro 3425</td><td> Gly</td><td> He Phe Arg Cys 3430</td><td> Asn</td><td> Gly Gin Asp Asn 3435</td><td> Cys</td><td> Gly 3440</td>

<td> Asp Gly Glu Asp</td><td> Glu</td><td> Arg Asp Cys Pro</td><td> Glu</td><td> Val Thr Cys Ala</td><td> Pro</td><td> Asn</td>

<td></td><td colspan="2"> 3445</td><td colspan="2"> 3450</td><td colspan="2"> 3455</td>

<td> Gin Phe Gin Cys</td><td> Ser</td><td> He Thr Lys Arg</td><td> Cys</td><td> He Pro Arg Val</td><td> Trp</td><td> Val</td>

<td colspan="2"> 3460</td><td colspan="2"> 3465</td><td colspan="2"> 3470</td><td></td>

<td> Cys Asp Arg Asp 3475</td><td> Asn</td><td> Asp Cys Val Asp 3480</td><td> Gly</td><td> Ser Asp Glu Pro 3485</td><td> Ala</td><td> Asn</td>

<td> Cys Thr Gin Met 3490</td><td> Thr</td><td> Cys Gly Val Asp 3495</td><td> Glu</td><td> Phe Arg Cys Lys 3500</td><td> Asp</td><td> Ser</td>

<td> Gly Arg Cys lie 3505</td><td> Pro</td><td> Ala Arg Trp Lys 3510</td><td> Cys</td><td> Asp Gly Glu Asp 3515</td><td> Asp</td><td> Cys 3520</td>

<td> Gly Asp Gly Ser</td><td> Asp</td><td> Glu Pro Lys Glu</td><td> Glu</td><td> Cys Asp Glu Arg</td><td> Thr</td><td> Cys</td>

3525 3530 3535

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<td rowspan="2"> Glu</td><td rowspan="2"> Pro</td><td colspan="2"> Tyr Gin Phe Arg Cys Lys Asn Asn Arg Cys Val</td><td rowspan="2"> Pro Gly Arg 3550</td>

<td> 3540</td><td> 3545</td>

<td> Trp</td><td> Gin</td><td> Cys Asp Tyr Asp Asn Asp</td><td> Cys Gly Asp Asn Ser</td><td> Asp Glu Glu</td>

<td></td><td></td><td colspan="3"> 3555 3560 356S</td>

<td> Ser</td><td> Cys</td><td> Thr Pro Arg Pro Cys Ser</td><td> Glu Ser Glu Phe Set</td><td> Cys Ala Asn</td>

<td></td><td colspan="2"> 3570 3575</td><td> 3580</td><td></td>

<td> Gly</td><td> Arg</td><td> Cys Ile Ala Gly Arg Trp</td><td> Lys Cys Asp Gly Asp</td><td> His Asp Cys</td>

<td colspan="2"> 3585</td><td> 3590</td><td> 3595</td><td> 3600</td>

<td> Ala</td><td> Asp</td><td> Gly Ser Asp Glu Lys Asp</td><td> Cys Thr Pro Arg Cys</td><td> Asp Met Asp</td>

<td></td><td></td><td> 3605</td><td> 3610</td><td> 3615</td>

<td> Gin</td><td> Phe</td><td> Gin Cys Lys Ser Gly His</td><td> Cys Ile Pro Leu Arg</td><td> Trp Arg Cys</td>

<td></td><td></td><td> 3620</td><td> 3625</td><td> 3630</td>

<td> Asp</td><td> Ala</td><td> Asp Ala Asp Cys Met Asp</td><td> Gly Ser Asp Glu Glu</td><td> Ala Cys Gly</td>

<td></td><td></td><td colspan="3"> 3635 3640 3645</td>

<td> Thr</td><td> Gly</td><td> Val Arg Thr Cys Pro Leu</td><td> Asp Glu Phe Gin Cys</td><td> Asn Asn Thr</td>

<td></td><td colspan="2"> 3650 3655</td><td> 3660</td><td></td>

<td> Leu</td><td> Cys</td><td> Lys Pro Leu Ala Trp Lys</td><td> Cys Asp Gly Glu Asp</td><td> Asp Cys Gly</td>

<td colspan="2"> 3665</td><td> 3670</td><td> 3675</td><td> 3680</td>

<td> Asp</td><td> Asn</td><td> Ser Asp Glu Asn Pro Glu</td><td> Glu Cys Ala Arg Phe</td><td> Val Cys Pro</td>

<td></td><td></td><td> 3685</td><td> 3690</td><td> 3695</td>

<td> Pro</td><td> Asn</td><td> Arg Pro Phe Arg Cys Lys</td><td> Asn Asp Arg Val Cys</td><td> Leu Trp Ile</td>

<td></td><td></td><td> 3700</td><td> 3705</td><td> 3710</td>

<td> Gly</td><td> Arg</td><td> Gin Cys Asp Gly Thr Asp</td><td> Asn Cys Gly Asp Gly</td><td> Thr Asp Glu</td>

<td></td><td></td><td colspan="3"> 3715 3720 3725</td>

<td> Glu</td><td> Asp</td><td> Cys Glu Pro Pro Thr Ala</td><td> His Thr Thr His Cys</td><td> Lys Asp Lys</td>

<td></td><td colspan="2"> 3730 3735</td><td> 3740</td><td></td>

<td> Lys</td><td> Glu</td><td> Phe Leu Cys Arg Asn Gin</td><td> Arg Cys Leu Ser Ser</td><td> Ser Leu Arg</td>

<td colspan="2"> 3745</td><td> 3750</td><td> 3755</td><td> 3760</td>

<td> Cys</td><td> Asn</td><td> Met Phe Asp Asp Cys Gly</td><td> Asp Gly Ser Asp Glu</td><td> Glu Asp Cys</td>

<td></td><td></td><td> 3765</td><td> 3770</td><td> 3775</td>

<td> Ser</td><td> Ile</td><td> Asp Pro Lys Leu Thr Ser</td><td> Cys Ala Thr Asn Ala</td><td> Ser Ile Cys</td>

<td></td><td></td><td> 3780</td><td> 3785</td><td> 3790</td>

<td> Gly</td><td> Asp</td><td> Glu Ala Arg Cys Val Arg</td><td> Thr Glu Lys Ala Ala</td><td> Tyr Cys Ala</td>

<td></td><td></td><td colspan="3"> 3795 3800 3805</td>

<td> Cys</td><td> Arg</td><td> Ser Gly Phe His Thr Val</td><td> Pro Gly Gin Pro Gly</td><td> Cys Gin Asp</td>

3810 3815 3820

Ile Asn Glu Cys Leu Arg Phe Gly Thr Cys Ser Gin Leu Cys Asn Asn 3325 3830 3835 3840

<img file="CA2155335C_D0030.tif" />

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<td> Thr</td><td> Lys Gly Gly</td><td> His Leu 3845</td><td> Cys</td><td> Ser</td><td> Cys Ala Arg 3850</td><td> Asn</td><td> Phe</td><td> Met</td><td> Lys Thr 3855</td>

<td> His</td><td> Asn Thr Cys</td><td> Lys Ala</td><td> Glu</td><td> Gly</td><td> Ser Glu Tyr</td><td> Gin</td><td> Val</td><td> Leu</td><td> Tyr He</td>

<td></td><td colspan="2"> 3860</td><td></td><td></td><td> 3865</td><td></td><td></td><td colspan="2"> 3870</td>

<td> Ala</td><td> Asp Asp Asn</td><td> Glu lie</td><td> Arg</td><td> Ser</td><td> Leu Phe Pro</td><td> Gly</td><td> His</td><td> Pro</td><td> His Ser</td>

<td></td><td> 3875</td><td></td><td></td><td colspan="2"> 3880</td><td></td><td colspan="2"> 3885</td><td></td>

<td> Ala</td><td> Tyr Glu Gin</td><td> Ala Phe</td><td> Gin</td><td> Gly</td><td> Asp Glu Ser</td><td> Val</td><td> Arg</td><td> He</td><td> Asp Ala</td>

<td></td><td> 3890</td><td></td><td colspan="2"> 3895</td><td></td><td colspan="2"> 3900</td><td></td><td></td>

<td> Met</td><td> Asp Val His</td><td> Val Lys</td><td> Ala</td><td> Gly</td><td> Arg Val Tyr</td><td> Trp</td><td> Thr</td><td> Asn</td><td> Trp His</td>

<td colspan="2"> 3905</td><td colspan="2"> 3910</td><td></td><td colspan="2"> 3915</td><td></td><td></td><td> 3920</td>

<td> Thr</td><td> Gly Thr lie</td><td> Ser Tyr</td><td> Arg</td><td> Ser</td><td> Leu Pro Pro</td><td> Ala</td><td> Ala</td><td> Pro</td><td> Pro Thr</td>

<td></td><td></td><td> 3925</td><td></td><td></td><td> 3930</td><td></td><td></td><td></td><td> 3935</td>

<td> Thr</td><td> Ser Asn Arg</td><td> His Arg</td><td> Arg</td><td> Gin</td><td> lie Asp Arg</td><td> Gly</td><td> Val</td><td> Thr</td><td> His Leu</td>

<td></td><td colspan="2"> 3940</td><td></td><td></td><td> 3945</td><td></td><td></td><td colspan="2"> 3950</td>

<td> Asn</td><td> lie Ser Gly</td><td> Leu Lys</td><td> Met</td><td> Pro</td><td> Arg Gly He</td><td> Ala</td><td> lie</td><td> Asp</td><td> Trp Val</td>

<td></td><td> 3955</td><td></td><td></td><td colspan="2"> 3960</td><td></td><td colspan="2"> 3965</td><td></td>

<td> Ala</td><td> Gly Asn Val</td><td> Tyr Trp</td><td> Thr</td><td> Asp</td><td> Ser Gly Arg</td><td> Asp</td><td> Val</td><td> He</td><td> Glu Val</td>

<td></td><td> 3970</td><td></td><td colspan="2"> 3975</td><td></td><td colspan="2"> 3980</td><td></td><td></td>

<td> Ala</td><td> Gin Met Lys</td><td> Gly Glu</td><td> Asn</td><td> Arg</td><td> Lys Thr Leu</td><td> He</td><td> Ser</td><td> Gly</td><td> Met He</td>

<td colspan="2"> 3985</td><td colspan="2"> 3990</td><td></td><td colspan="2"> 3995</td><td></td><td></td><td> 4000</td>

<td> Asp</td><td> Glu Pro His</td><td> Ala He</td><td> Val</td><td> Val</td><td> Asp Pro Leu</td><td> Arg</td><td> Gly</td><td> Thr</td><td> Met Tyr</td>

<td></td><td></td><td> 4005</td><td></td><td></td><td> 4010</td><td></td><td></td><td></td><td> 4015</td>

<td> Trp</td><td> Ser Asp Trp</td><td> Gly Asn</td><td> His</td><td> Pro</td><td> Lys He Glu</td><td> Thr</td><td> Ala</td><td> Ala</td><td> Met Asp</td>

<td></td><td colspan="2"> 4020</td><td></td><td></td><td> 4025</td><td></td><td></td><td colspan="2"> 4030</td>

<td> Gly</td><td> Thr Leu Arg</td><td> Glu Thr</td><td> Leu</td><td> Val</td><td> Gin Asp Asn</td><td> He</td><td> Gin</td><td> Trp</td><td> Pro Thr</td>

<td></td><td> 4035</td><td></td><td></td><td colspan="2"> 4040</td><td></td><td colspan="2"> 4045</td><td></td>

<td> Gly</td><td> Leu Ala Val</td><td> Asp Tyr</td><td> His</td><td> Asn</td><td> Glu Arg Leu</td><td> Tyr</td><td> Trp</td><td> Ala</td><td> Asp Ala</td>

<td></td><td> 4050</td><td></td><td colspan="2"> 4055</td><td></td><td colspan="2"> 4060</td><td></td><td></td>

<td> Lys</td><td> Leu Ser Val</td><td> He Gly</td><td> Ser</td><td> He</td><td> Arg Leu Asn</td><td> Gly</td><td> Thr</td><td> Asp</td><td> Pro lie</td>

<td colspan="2"> 4065</td><td colspan="2"> 4070</td><td></td><td colspan="2"> 4075</td><td></td><td></td><td> 4080</td>

<td> Val</td><td> Ala Ala Asp</td><td> Ser Lys</td><td> Arg</td><td> Gly</td><td> Leu Ser His</td><td> Pro</td><td> Phe</td><td> Ser</td><td> He Asp</td>

<td></td><td></td><td> 4085</td><td></td><td></td><td> 4090</td><td></td><td></td><td></td><td> 4095</td>

<td> Val</td><td> Phe Glu Asp</td><td> Tyr He</td><td> Tyr</td><td> Gly</td><td> Val Thr Tyr</td><td> lie</td><td> Asn</td><td> Asn</td><td> Arg Val</td>

<td></td><td colspan="2"> 4100</td><td></td><td></td><td> 4105</td><td></td><td></td><td colspan="2"> 4110</td>

<td> Phe</td><td> Lys lie His</td><td> Lys Phe</td><td> Gly</td><td> His</td><td> Ser Pro Leu</td><td> Val</td><td> Asn</td><td> Leu</td><td> Thr Gly</td>

4115 4120 4125

Gly Leu Ser His Ala Ser Asp Val Val Leu Tyr His Gin His Lys Gin 4130 4135 4140

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<td> Pro Glu 4145</td><td> Val</td><td> Thr</td><td> Asn Pro Cys 4150</td><td> Asp Arg</td><td> Lys Lys Cys 4155</td><td> Glu</td><td> Trp Leu</td><td> Cys 4160</td>

<td> Leu Leu</td><td> Ser</td><td> Pro</td><td> Ser Gly Pro</td><td> Val Cys</td><td> Thr Cys Pro</td><td> Asn</td><td> Gly Lys</td><td> Arg</td>

<td></td><td></td><td></td><td> 4165</td><td></td><td> 4170</td><td></td><td colspan="2"> 4175</td>

<td> Leu Asp</td><td> Asn</td><td> Gly</td><td> Thr Cys Val</td><td> Pro Val</td><td> Pro Ser Pro</td><td> Thr</td><td> Pro Pro</td><td> Pro</td>

<td></td><td></td><td colspan="2"> 4180</td><td colspan="2"> 4185</td><td></td><td> 4190</td><td></td>

<td> Asp Ala</td><td> Pro</td><td> Arg</td><td> Pro Gly Thr</td><td> Cys Asn</td><td> Leu Gin Cys</td><td> Phe</td><td> Asn Gly</td><td> Gly</td>

<td></td><td colspan="2"> 4195</td><td></td><td> 4200</td><td></td><td colspan="2"> 4205</td><td></td>

<td> Ser Cys</td><td> Phe</td><td> Leu</td><td> Asn Ala Arg</td><td> Arg Gin</td><td> Pro Lys Cys</td><td> Arg</td><td> Cys Gin</td><td> Pro</td>

<td colspan="2"> 4210</td><td></td><td colspan="2"> 4215</td><td colspan="2"> 4220</td><td></td><td></td>

<td> Arg Tyr</td><td> Thr</td><td> Gly</td><td> Asp Lys Cys</td><td> Glu Leu</td><td> Asp Gin Cys</td><td> Trp</td><td> Glu His</td><td> Cys</td>

<td> 4225</td><td></td><td></td><td> 4230</td><td></td><td> 4235</td><td></td><td></td><td> 4240</td>

<td> Arg Asn</td><td> Gly</td><td> Gly</td><td> Thr Cys Ala</td><td> Ala Ser</td><td> Pro Ser Gly</td><td> Met</td><td> Pro Thr</td><td> Cys</td>

<td></td><td></td><td></td><td> 4245</td><td></td><td> 4250</td><td></td><td colspan="2"> 4255</td>

<td> Arg Cys</td><td> Pro</td><td> Thr</td><td> Gly Phe Thr</td><td> Gly Pro</td><td> Lys Cys Thr</td><td> Gin</td><td> Gin Val</td><td> Cys</td>

<td></td><td></td><td colspan="2"> 4260</td><td colspan="2"> 4265</td><td></td><td> 4270</td><td></td>

<td> Ala Gly</td><td> Tyr</td><td> Cys</td><td> Ala Asn Asn</td><td> Ser Thr</td><td> Cys Thr Val</td><td> Asn</td><td> Gin Gly</td><td> Asn</td>

<td></td><td colspan="2"> 4275</td><td></td><td> 4280</td><td></td><td colspan="2"> 4285</td><td></td>

<td> Gin Pro</td><td> Gin</td><td> Cys</td><td> Arg Cys Leu</td><td> Pro Gly</td><td> Phe Leu Gly</td><td> Asp</td><td> Arg Cys</td><td> Gin</td>

<td colspan="2"> 4290</td><td></td><td colspan="2"> 4295</td><td colspan="2"> 4300</td><td></td><td></td>

<td> Tyr Arg</td><td> Gin</td><td> Cys</td><td> Ser Gly Tyr</td><td> Cys Glu</td><td> Asn Phe Gly</td><td> Thr</td><td> Cys Gin</td><td> Met</td>

<td> 4305</td><td></td><td></td><td> 4310</td><td></td><td> 4315</td><td></td><td></td><td> 4320</td>

<td> Ala Ala</td><td> Asp</td><td> Gly</td><td> Ser Arg Gin</td><td> Cys Arg</td><td> Cys Thr Ala</td><td> Tyr</td><td> Phe Glu</td><td> Gly</td>

<td></td><td></td><td></td><td> 4325</td><td></td><td> 4330</td><td></td><td colspan="2"> 4335</td>

<td> Ser Arg</td><td> Cys</td><td> Glu</td><td> Val Asn Lys</td><td> Cys Ser</td><td> Arg Cys Leu</td><td> Glu</td><td> Gly Ala</td><td> Cys</td>

<td></td><td></td><td colspan="2"> 4340</td><td colspan="2"> 4345</td><td></td><td> 4350</td><td></td>

<td> Val Val</td><td> Asn</td><td> Lys</td><td> Gin Ser Gly</td><td> Asp Val</td><td> Thr Cys Asn</td><td> Cys</td><td> Thr Asp</td><td> Gly</td>

<td></td><td colspan="2"> 4355</td><td></td><td> 4360</td><td></td><td colspan="2"> 4365</td><td></td>

<td> Arg Val</td><td> Ala</td><td> Pro</td><td> Ser Cys Leu</td><td> Thr Cys</td><td> Val Gly His</td><td> Cys</td><td> Ser Asn</td><td> Gly</td>

<td colspan="2"> 4370</td><td></td><td colspan="2"> 4375</td><td colspan="2"> 4380</td><td></td><td></td>

<td> Gly Ser</td><td> Cys</td><td> Thr</td><td> Met Asn Ser</td><td> Lys Met</td><td> Met Pro Glu</td><td> Cys</td><td> Gin Cys</td><td> Pro</td>

<td> 4385</td><td></td><td></td><td> 4390</td><td></td><td> 4395</td><td></td><td></td><td> 4400</td>

<td> Pro His</td><td> Met</td><td> Thr</td><td> Gly Pro Arg</td><td> Cys Glu</td><td> Glu His Val</td><td> Phe</td><td> Ser Gin</td><td> Gin</td>

<td></td><td></td><td></td><td> 4405</td><td></td><td> 4410</td><td></td><td colspan="2"> 4415</td>

<td> Gin Pro</td><td> Gly</td><td> His</td><td> He Ala Ser</td><td> He Leu</td><td> He Pro Leu</td><td> Leu</td><td> Leu Leu</td><td> Leu</td>

4420 4425 4430

Leu Leu Val Leu Val Ala Gly Val Val Phe Trp Tyr Lys Arg Arg Val 4435 4440 4445

<img file="CA2155335C_D0031.tif" />

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<img file="CA2155335C_D0032.tif" />

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Gin Gly Ala 4450

Asn Val Glu 4465

Pro Asp Asp

Asp Lys Pro

Gly Gly His 451‘

Glu Leu Leu 4530

<td> Lys Gly</td><td> Phe Gin His 4455</td><td> Gin Arg Met Thr Asn Gly Ala 4460</td><td> Met</td>

<td> He Gly</td><td> Asn Pro Thr 4470</td><td> Tyr Lys Met Tyr Glu Gly Gly 4475</td><td> Glu 4480</td>

<td colspan="2"> Val Gly Gly Leu Leu 4485</td><td colspan="2"> Asp Ala Asp Phe Ala Leu Asp Pro 4490 4495</td>

<td> Thr Asn 4500</td><td> Phe Thr Asn</td><td> Pro Val Tyr Ala Thr Leu Tyr 4505 4510</td><td> Met</td>

<td> Gly Ser</td><td colspan="2"> Arg His Ser Leu Ala Ser Thr Asp Glu Lys 4520 4525</td><td> Arg</td>

<td> Gly Arg</td><td colspan="2"> Gly Pro Glu Asp Glu He Gly Asp Pro Leu</td><td> Ala</td>

4535 4540

C2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 487 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

<td> Met 1</td><td> Ala</td><td> Gly</td><td> Leu</td><td> Leu 5</td><td> His</td><td> Leu</td><td> Val</td><td> Leu</td><td> Leu 10</td><td> Ser</td><td> Thr</td><td> Ala</td><td> Leu</td><td> Gly 15</td><td> Gly</td>

<td> Leu</td><td> Leu</td><td> Arg</td><td> Pro 20</td><td> Ala</td><td> Gly</td><td> Ser</td><td> Val</td><td> Phe 25</td><td> Leu</td><td> Pro</td><td> Arg</td><td> Asp</td><td> Gin 30</td><td> Ala</td><td> His</td>

<td> Arg</td><td> Val</td><td> Leu 35</td><td> Gin</td><td> Arg</td><td> Ala</td><td> Arg</td><td> Arg 40</td><td> Ala</td><td> Asn</td><td> Ser</td><td> Phe</td><td> Leu 45</td><td> Glu</td><td> Glu</td><td> Val</td>

<td> Lys</td><td> Gin 50</td><td> Gly</td><td> Asn</td><td> Leu</td><td> Glu</td><td> Arg 55</td><td> Glu</td><td> Cys</td><td> Leu</td><td> Glu</td><td> Glu 60</td><td> Ala</td><td> Cys</td><td> Ser</td><td> Leu</td>

<td> Glu 65</td><td> Glu</td><td> Ala</td><td> Arg</td><td> Glu</td><td> Val 70</td><td> Phe</td><td> Glu</td><td> Asp</td><td> Ala</td><td> Glu 75</td><td> Gin</td><td> Thr</td><td> Asp</td><td> Glu</td><td> Phe 80</td>

<td> Trp</td><td> Ser</td><td> Lys</td><td> Tyr</td><td> Lys 85</td><td> Asp</td><td> Gly</td><td> Asp</td><td> Gin</td><td> Cys 90</td><td> Glu</td><td> Gly</td><td> His</td><td> Pro</td><td> Cys 95</td><td> Leu</td>

<td> Asn</td><td> Gin</td><td> Gly</td><td> His 100</td><td> Cys</td><td> Lys</td><td> Asp</td><td> Gly</td><td> He 105</td><td> Gly</td><td> Asp</td><td> Tyr</td><td> Thr</td><td> Cys 110</td><td> Thr</td><td> Cys</td>

<td> Ala</td><td> Glu</td><td> Gly 115</td><td> Phe</td><td> Glu</td><td> Gly</td><td> Lys</td><td> Asn 120</td><td> Cys</td><td> Glu</td><td> Phe</td><td> Ser</td><td> Thr 125</td><td> Arg</td><td> Glu</td><td> He</td>

<td> Cys</td><td> Ser 130</td><td> Leu</td><td> Asp</td><td> Asn</td><td> Gly</td><td> Gly 135</td><td> Cys</td><td> Asp</td><td> Gin</td><td> Phe</td><td> Cys 140</td><td> Arg</td><td> Glu</td><td> Glu</td><td> Arg</td>

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Ser Glu Val Arg Cys Ser Cys 145 150

Ser Lys Ser Cys Val Ser Thr 165

Gin Gly Arg Ser Arg Arg Trp

1B0

Asp Ala Ser Glu Leu Glu His 195

Glu Ser Ser Leu Asp Leu Leu 210 215

Gly Glu Asp Gly Ser Gin Val 225 230

Ala Glu Gly Glu Cys Pro Trp 245

Glu Gly Phe Cys Gly Gly Thr 260

Ala Ala His Cys Leu His Gin 275

Asp Arg Asn Thr Glu Gin Glu 290 295

Glu Met Thr Val Lys His Ser 305 310

Asp lie Ala Val Leu Arg Leu 325

Val Ala Pro Ala Cys Leu Pro 340

Met Thr Gin Lÿs Thr Gly lie 355

Lys Gly Arg Leu Ser Ser Thr 370 375

Asp Arg Ser Thr Cys Lys Leu 385 390

Met Phe Cys Ala Gly Tyr Asp 405

Asp Ser Gly Gly Pro His Val 420

<td> His</td><td> Gly</td><td> Tyr 155</td><td> Val</td><td> Leu</td><td> Gly</td><td> Asp</td><td> Asp 160</td>

<td> Arg</td><td> Phe 170</td><td> Pro</td><td> Cys</td><td> Gly</td><td> Lys</td><td> Phe 175</td><td> Thr</td>

<td> lie 185</td><td> His</td><td> Thr</td><td> Ser</td><td> Glu</td><td> Asp 190</td><td> Ala</td><td> Leu</td>

<td> Asp</td><td> Pro</td><td> Ala</td><td> Asp</td><td> Leu 205</td><td> Ser</td><td> Pro</td><td> Thr</td>

<td> Leu</td><td> Asn</td><td> Arg</td><td> Thr 220</td><td> Glu</td><td> Pro</td><td> Ser</td><td> Ala</td>

<td> Arg</td><td> He</td><td> Val 235</td><td> Gly</td><td> Gly</td><td> Arg</td><td> Asp</td><td> Cys 240</td>

<td> Ala</td><td> Leu 250</td><td> Leu</td><td> Val</td><td> Asn</td><td> Glu</td><td> Glu 255</td><td> Asn</td>

<td> Leu 265</td><td> Asn</td><td> Glu</td><td> Phe</td><td> Tyr</td><td> Val 270</td><td> Leu</td><td> Thr</td>

<td> Lys</td><td> Arg</td><td> Phe</td><td> Thr</td><td> Val 285</td><td> Arg</td><td> Val</td><td> Gly</td>

<td> Gly</td><td> Asn</td><td> Glu</td><td> Met 300</td><td> Ala</td><td> His</td><td> Glu</td><td> Val</td>

<td> Phe</td><td> Val</td><td> Lys 315</td><td> Glu</td><td> Thr</td><td> Tyr</td><td> Asp</td><td> Phe 320</td>

<td> Thr</td><td> Pro 330</td><td> He</td><td> Arg</td><td> Phe</td><td> Arg</td><td> Arg 335</td><td> Asn</td>

<td> Lys 345</td><td> Asp</td><td> Trp</td><td> Ala</td><td> Glu</td><td> Ala 350</td><td> Thr</td><td> Leu</td>

<td> Ser</td><td> Gly</td><td> Phe</td><td> Gly</td><td> Arg 365</td><td> Thr</td><td> His</td><td> Glu</td>

<td> Lys</td><td> Met</td><td> Leu</td><td> Glu 380</td><td> Val</td><td> Pro</td><td> Tyr</td><td> Val</td>

<td> Ser</td><td> Ser</td><td> Phe 395</td><td> Thr</td><td> He</td><td> Thr</td><td> Pro</td><td> Asn 400</td>

<td> Gin</td><td> Pro 410</td><td> Glu</td><td> Asp</td><td> Ala</td><td> Cys</td><td> Gin 415</td><td> Gly</td>

<td> Arg 425</td><td> Phe</td><td> Lys</td><td> Asp</td><td> Thr</td><td> Tyr 430</td><td> Phe</td><td> Val</td>

Thr Gly He Val Ser Trp Gly Glu Gly Cys Ala Arg Lys Gly Lys Phe 435 440 445

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<td colspan="2"> Gly Val</td><td rowspan="2"> Tyr Thr</td><td rowspan="2"> Lys Val</td><td rowspan="2"> Ser Asn Phe 455</td><td rowspan="2"> Leu Lys Trp 460</td><td rowspan="2"> He Asp</td><td rowspan="2"> Lys</td><td rowspan="2"> He</td>

<td></td><td> 450</td>

<td> Met</td><td> Lys</td><td> Ala Arg</td><td> Ala Gly</td><td> Ala Ala Gly</td><td> Ser Arg Gly</td><td> His Ser</td><td> Glu</td><td> Ala</td>

<td> 465</td><td></td><td></td><td> 470</td><td></td><td> 475</td><td></td><td></td><td> 480</td>

<td> Pro</td><td> A j. a</td><td> Thr Trp</td><td> Thr Val</td><td> Pro</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="5"> INFORMATION FOR SEQ ID NO: 54:</td><td></td><td></td><td></td><td></td>

<td> (i)</td><td colspan="4"> SEQUENCE CHARACTERISTICS:</td><td></td><td></td><td></td><td></td>

<td></td><td> (A)</td><td> LENGTH</td><td colspan="2"> : 790 amino acids</td><td></td><td></td><td></td><td></td>

<td></td><td> (B)</td><td colspan="3"> TYPE : amino acid</td><td></td><td></td><td></td><td></td>

<td></td><td> (C)</td><td colspan="3"> STRANDEDNESS: single</td><td></td><td></td><td></td><td></td>

<td></td><td> (D)</td><td colspan="3"> TOPOLOGY: linear</td><td></td><td></td><td></td><td></td>

<td> (ii&gt;</td><td colspan="4"> MOLECULE TYPE: protein</td><td></td><td></td><td></td><td></td>

<td> (xiï</td><td colspan="4"> SEQUENCE DESCRIPTION: SEQ ID NO:</td><td> : 54:</td><td></td><td></td><td></td>

<td> Glu</td><td> Pro</td><td> Leu Asp</td><td> Asp Tyr</td><td> Val Asn Thr</td><td> Gin Gly Ala</td><td> Ser Leu</td><td> Phe</td><td> Ser</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td> 10</td><td></td><td> 15</td><td></td>

<td> Val</td><td> Thr</td><td> Lys Lys</td><td> Gin Leu</td><td> Gly Ala Gly</td><td> Ser He Glu</td><td> Glu Cys</td><td> Ala</td><td> Ala</td>

<td></td><td></td><td> 20</td><td></td><td> 25</td><td></td><td> 30</td><td></td><td></td>

<td> Lys</td><td> Cys</td><td> Glu Glu</td><td> Asp Glu</td><td> Glu Phe Thr</td><td> Cys Arg Ala</td><td> Phe Gin</td><td> Tyr</td><td> His</td>

<td></td><td></td><td> 35</td><td></td><td> 40</td><td></td><td> 45</td><td></td><td></td>

<td> Ser</td><td> Lys</td><td> Glu din</td><td> Gin Cys</td><td> Val He Met</td><td> Ala Glu Asn</td><td> Arg Lys</td><td> Ser</td><td> Ser</td>

<td></td><td> 50</td><td></td><td></td><td> 55</td><td> 60</td><td></td><td></td><td></td>

<td> He</td><td> He</td><td> Arg Met</td><td> Arg Asp</td><td> Val Val Leu</td><td> Phe Glu Lys</td><td> Lys Val</td><td> Tyr</td><td> Leu</td>

<td> 65</td><td></td><td></td><td> 70</td><td></td><td> 75</td><td></td><td></td><td> 80</td>

<td> Ser</td><td> Glu</td><td> Cys Lys</td><td> Thr Gly</td><td> Asn Gly Lys</td><td> Asn Tyr Arg</td><td> Gly Thr</td><td> Met</td><td> Ser</td>

<td></td><td></td><td></td><td> 85</td><td></td><td> 90</td><td></td><td> 95</td><td></td>

<td> Lys</td><td> Thr</td><td> Lys Asn</td><td> Gly He</td><td> Thr Cys Gin</td><td> Lys Trp Ser</td><td> Ser Thr</td><td> Ser</td><td> Pro</td>

<td></td><td></td><td> 100</td><td></td><td> 105</td><td></td><td> 110</td><td></td><td></td>

<td> His</td><td> Arg</td><td> Pro Arg</td><td> Phe Ser</td><td> Pro Ala Thr</td><td> His Pro Ser</td><td> Glu Gly</td><td> Leu</td><td> Glu</td>

<td></td><td></td><td> 115</td><td></td><td> 120</td><td></td><td> 125</td><td></td><td></td>

<td> Glu</td><td> Asn</td><td> Tyr Cys</td><td> Arg Asn</td><td> Pro Asp Asn</td><td> Asp Pro Gin</td><td> Gly Pro</td><td> Trp</td><td> Cys</td>

<td></td><td> 130</td><td></td><td></td><td> 135</td><td> 140</td><td></td><td></td><td></td>

<td> Tyr</td><td> Thr</td><td> Thr Asp</td><td> Pro Glu</td><td> Lys Arg Tyr</td><td> Asp Tyr Cys</td><td> Asp He</td><td> Leu</td><td> Glu</td>

<td> 145</td><td></td><td></td><td> 150</td><td></td><td> 155</td><td></td><td></td><td> 160</td>

<td> Cys</td><td> Glu</td><td> Glu Glu</td><td> Cys Met</td><td> His Cys Ser</td><td> Gly Glu Asn</td><td> Tyr Asp</td><td> Gly</td><td> Lys</td>

165 170 175

Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gin Ala Trp Asp Ser Gin 180 185 190

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<td> Ser</td><td> Pro</td><td> His 195</td><td> Ala</td><td> His</td><td> Gly</td><td> Tyr</td><td> He 200</td><td> Pro</td><td> Ser</td><td> Lys</td><td> Phe</td><td> Pro 205</td><td> Asn</td><td> Lys</td><td> Asn</td>

<td> Leu</td><td> Lys 210</td><td> Lys</td><td> Asn</td><td> Tyr</td><td> Cys</td><td> Arg 215</td><td> Asn</td><td> Pro</td><td> Asp</td><td> Arg</td><td> Glu 220</td><td> Leu</td><td> Arg</td><td> Pro</td><td> Trp</td>

<td> Cys 225</td><td> Phe</td><td> Thr</td><td> Thr</td><td> Asp</td><td> Pro 230</td><td> Asn</td><td> Lys</td><td> Arg</td><td> Trp</td><td> Glu 235</td><td> Leu</td><td> Cys</td><td> Asp</td><td> lie</td><td> Pro 240</td>

<td> Arg</td><td> Cys</td><td> Thr</td><td> Thr</td><td> Pro 245</td><td> Pro</td><td> Pro</td><td> Ser</td><td> Ser</td><td> Gly 250</td><td> Pro</td><td> Thr</td><td> Tyr</td><td> Gin</td><td> Cys 255</td><td> Leu</td>

<td> Lys</td><td> Gly</td><td> Thr</td><td> Gly 260</td><td> Glu</td><td> Asn</td><td> Ty_r</td><td> Arg</td><td> Gly 265</td><td> Asn</td><td> Val</td><td> Ala</td><td> Val</td><td> Thr 270</td><td> Val</td><td> Ser</td>

<td> Gly</td><td> His</td><td> Thr 275</td><td> Cys</td><td> Gin</td><td> His</td><td> Trp</td><td> Ser 280</td><td> Ala</td><td> Gin</td><td> Thr</td><td> Pro</td><td> His 285</td><td> Thr</td><td> His</td><td> Asn</td>

<td> Arg</td><td> Thr 290</td><td> Pro</td><td> Glu</td><td> Asn</td><td> Phe</td><td> Pro 295</td><td> Cys</td><td> Lys</td><td> Asn</td><td> Leu</td><td> Asp 300</td><td> Glu</td><td> Asn</td><td> Tyr</td><td> Cys</td>

<td> Arg 305</td><td> Asn</td><td> Pro</td><td> Asp</td><td> Gly</td><td> Lys 310</td><td> Arg</td><td> Ala</td><td> Pro</td><td> Trp</td><td> Cys 315</td><td> His</td><td> Thr</td><td> Thr</td><td> Asn</td><td> Ser 320</td>

<td> Gin</td><td> Val</td><td> Arg</td><td> Trp</td><td> Glu 325</td><td> Tyr</td><td> Cys</td><td> Lys</td><td> He</td><td> Pro 330</td><td> Ser</td><td> Cys</td><td> Asp</td><td> Ser</td><td> Ser 335</td><td> Pro</td>

<td> Val</td><td> Ser</td><td> Thr</td><td> Glu 340</td><td> Glu</td><td> Leu</td><td> Ala</td><td> Pro</td><td> Thr 345</td><td> Ala</td><td> Pro</td><td> Pro</td><td> Glu</td><td> Leu 350</td><td> Thr</td><td> Pro</td>

<td> Val</td><td> Val</td><td> Gin 355</td><td> Asp</td><td> Cys</td><td> Tyr</td><td> His</td><td> Gly 360</td><td> Asp</td><td> Gly</td><td> Gin</td><td> Ser</td><td> Tyr 365</td><td> Arg</td><td> Gly</td><td> Çhr</td>

<td> Ser</td><td> Ser 370</td><td> Thr</td><td> Thr</td><td> Thr</td><td> Thr</td><td> Gly 375</td><td> Lys</td><td> Lys</td><td> Cys</td><td> Gin</td><td> Ser 380</td><td> Trp</td><td> Ser</td><td> Ser</td><td> Met</td>

<td> Thr 385</td><td> Pro</td><td> His</td><td> Arg</td><td> His</td><td> Gin 390</td><td> Lys</td><td> Thr</td><td> Pro</td><td> Glu</td><td> Asn 395</td><td> Tyr</td><td> Pro</td><td> Asn</td><td> Ala</td><td> Gly 400</td>

<td> Leu</td><td> Thr</td><td> Met</td><td> Asn</td><td> Tyr 405</td><td> Cys</td><td> Arg</td><td> Asn</td><td> Pro</td><td> Asp 410</td><td> Ala</td><td> Asp</td><td> Lys</td><td> Gly</td><td> Pro 415</td><td> Trp</td>

<td> Cys</td><td> Phe</td><td> Thr</td><td> Thr 420</td><td> Asp</td><td> Pro</td><td> Ser</td><td> Val</td><td> Arg 425</td><td> Trp</td><td> Glu</td><td> Tyr</td><td> Cys</td><td> Asn 430</td><td> Leu</td><td> Lys</td>

<td> Lys</td><td> Cys</td><td> Ser 435</td><td> Gly</td><td> Thr</td><td> Glu</td><td> Ala</td><td> Ser 440</td><td> Val</td><td> Val</td><td> Ala</td><td> Pro</td><td> Pro 445</td><td> Pro</td><td> Val</td><td> Val</td>

<td> Leu</td><td> Leu 450</td><td> Pro</td><td> Asn</td><td> Val</td><td> Glu</td><td> Thr 455</td><td> Pro</td><td> Ser</td><td> Glu</td><td> Glu</td><td> Asp 460</td><td> Cys</td><td> Met</td><td> Phe</td><td> Gly</td>

<td> Asn 465</td><td> Gly</td><td> Lys</td><td> Gly</td><td> Tyr</td><td> Arg 470</td><td> Gly</td><td> Lys</td><td> Arg</td><td> Ala</td><td> Thr 475</td><td> Thr</td><td> Val</td><td> Thr</td><td> Gly</td><td> Thr 480</td>

<td> Pro</td><td> Cys</td><td> Gin</td><td> Asp</td><td> Trp 485</td><td> Ala</td><td> Ala</td><td> Gin</td><td> Glu</td><td> Pro 490</td><td> His</td><td> Arg</td><td> His</td><td> Ser</td><td> He 495</td><td> Phe</td>

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150

Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg 500 505 510

Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro 515 520 525

Arg Lys Leu Tyr Asp Tyr Cys Asp val Pro Gin Cys Ala Ala Pro Ser 530 535 540

Phe Asp Cys Gly Lys Pro Gin Val Glu Pro Lys Lys Cys Pro Gly Arg

545 550 555 560

Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gin Val

565 570 575

Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu He 580 585 590

Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro 595 600 605

Arg Pro Ser Ser Tyr Lys Val lie Leu Gly Ala His Gin Glu Val Asn 610 615 620

Leu Glu Pro His Val Gin Glu He Glu Val Ser Arg Leu Phe Leu Glu

625 630 635 640

Pro Thr Arg Lys Asp He Ala Leu Leu Lys Leu Ser Ser Pro Ala Val

645 650 655 lie Thr Asp Lys Val He Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val 660 665 670

Val Ala Asp Arg Thr Glu Cys Phe He Thr Gly Trp Gly Glu Thr Gin 675 680 685

Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gin Leu Pro Val He 690 695 700

Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gin

705 710 715 720

Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys

725 730 735

Gin Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr 740 745 750 lie Leu Gin Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn 755 760 765

Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp He Glu 770 775 780

Gly Val Met Arg Asn Asn 785 790

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151 (2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 153 amino acids (Bl TYPE: amino acid (CÏ STRANDEDNESS : single (D/ TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

Val Tyr Leu Gin Thr Ser Leu Lys Tyr Asn He Leu Pro Glu Lys Glu 15 10 15

Glu Phe Pro Phe Ala Leu Gly Val Gin Thr Leu Pro Gin Thr Cys Asp 20 25 30

Glu Pro Lys Ala His Thr Ser Phe Gin He Ser Leu Ser Val Ser Tyr 35 40 45

Thr Gly Ser Arg Ser Ala Ser Asn Met Ala He Val Asp Val Lys Met 50 55 60

Val Ser Gly Phe lie Pro Leu Lys Pro Thr Val Lys Met Leu Glu Arg

70 75 80

Ser Asn His Val Ser Arg Thr Glu Val Ser Ser Asn His Val Leu lie

90 95

Tyr Leu Asp Lys Val Ser Asn Gin Thr Leu Ser Leu Phe Phe Thr Val 100 105 110

Leu Gin Asp Val Pro Val Arg Asp Leu Lys Pro Ala He Val Lys Val 115 120 125

Tyr Asp Tyr Tyr Glu Thr Asp Glu Phe Ala He Ala Glu Tyr Asn Ala 130 135 140

Pro Cys Ser Lys Asp Leu Gly Asn Ala 145 150 (2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 202 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

Met Glu Leu Trp Gly Ala Tyr Leu Leu Leu Cys Leu Phe Ser Leu Leu 15 10 15

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152

<td> Thr</td><td> Gin</td><td> Val</td><td> Thr 20</td><td> Thr</td><td> Glu</td><td> Pro</td><td> Pro</td><td> Thr 25</td><td> Gin</td><td> Lys</td><td> Pro</td><td> Lys</td><td> Lys 30</td><td> He</td><td> Val</td>

<td> Asn</td><td> Ala</td><td> Lys 35</td><td> Lys</td><td> Asp</td><td> Val</td><td> Val</td><td> Asn 40</td><td> Thr</td><td> Lys</td><td> Met</td><td> Phe</td><td> Glu 45</td><td> Glu</td><td> Leu</td><td> Lys</td>

<td> Ser</td><td> Arg 50</td><td> Leu</td><td> Asp</td><td> Thr</td><td> Leu</td><td> Ala 55</td><td> Gin</td><td> Glu</td><td> Val</td><td> Ala</td><td> Leu 60</td><td> Leu</td><td> Lys</td><td> Ga.U</td><td> Gin</td>

<td> Gin 65</td><td> Ala</td><td> Leu</td><td> Gin</td><td> Thr</td><td> Val 70</td><td> Cys</td><td> Leu</td><td> Lys</td><td> Gly</td><td> Thr 75</td><td> Lys</td><td> Val</td><td> His</td><td> Met</td><td> Lys 80</td>

<td> Cys</td><td> Phe</td><td> Leu</td><td> Ala</td><td> Phe 85</td><td> Thr</td><td> Gin</td><td> Thr</td><td> Lys</td><td> Thr 90</td><td> Phe</td><td> His</td><td> Glu</td><td> Ala</td><td> Ser 95</td><td> Glu</td>

<td> Asp</td><td> Cys</td><td> He</td><td> Ser 100</td><td> Arg</td><td> Gly</td><td> Gly</td><td> Thr</td><td> Leu 105</td><td> Ser</td><td> Thr</td><td> Pro</td><td> Gin</td><td> Thr 110</td><td> Gly</td><td> Ser</td>

<td> Glu</td><td> Asn</td><td> Asp 115</td><td> Ala</td><td> Leu</td><td> Tyr</td><td> Glu</td><td> Tyr 120</td><td> Leu</td><td> Arg</td><td> Gin</td><td> Ser</td><td> Val 125</td><td> Gly</td><td> Asn</td><td> Glu</td>

<td> Ala</td><td> Glu 130</td><td> He</td><td> Trp</td><td> Leu</td><td> Gly</td><td> Leu 135</td><td> Asn</td><td> Asp</td><td> Met</td><td> Ala</td><td> Ala 140</td><td> Glu</td><td> Gly</td><td> Thr</td><td> Trp</td>

<td> Val 145</td><td> Asp</td><td> Met</td><td> Thr</td><td> Gly</td><td> Ala 150</td><td> Arg</td><td> He</td><td> Ala</td><td> Tyr</td><td> Lys 155</td><td> Asn</td><td> Trp</td><td> Glu</td><td> Thr</td><td> Glu 160</td>

<td> He</td><td> Thr</td><td> Ala</td><td> Gin</td><td> Pro 165</td><td> Asp</td><td> Gly</td><td> Gly</td><td> Lys</td><td> Thr 170</td><td> Glu</td><td> Asn</td><td> Cys</td><td> Ala</td><td> Val 175</td><td> Leu</td>

<td> Ser</td><td> Gly</td><td> Ala</td><td> Ala 180</td><td> Asn</td><td> Gly</td><td> Lys</td><td> Trp</td><td> Phe 185</td><td> Asp</td><td> Lys</td><td> Arg</td><td> Cys</td><td> Arg 190</td><td> Asp</td><td> Gin</td>

<td> Leu</td><td> Pro</td><td> Tyr 195</td><td> He</td><td> Cys</td><td> Gin</td><td> Phe</td><td> Gly 200</td><td> He</td><td> Val</td><td></td><td></td><td></td><td></td><td></td><td></td>

(2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 246 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:

<td> Gin</td><td> Val</td><td> Lys</td><td> Leu</td><td> Gin</td><td> Gin</td><td> Ser</td><td> Gly</td><td> Ala</td><td> Glu</td><td> Leu</td><td> Val</td><td> Lys</td><td> Pro</td><td> Gly</td><td> Ala</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td> Ser</td><td> Val</td><td> Lys</td><td> Met</td><td> Ser</td><td> Cys</td><td> Lys</td><td> Ala</td><td> Ser</td><td> Gly</td><td> Tyr</td><td> Thr</td><td> Phe</td><td> Ala</td><td> Ser</td><td> Tyr</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

Trp He Asn Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He 35 40 45

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<td colspan="2"> Gly His</td><td rowspan="2"> He</td><td rowspan="2"> Tyr</td><td rowspan="2"> Pro</td><td colspan="10"> Val Arg Ser He Thr Lys Tyr Asn Glu Lys</td><td rowspan="2"> Phe</td>

<td></td><td> 50</td><td colspan="2"> 55</td><td colspan="8"> 60</td>

<td> Lys</td><td> Ser</td><td> Lys</td><td> Ala</td><td> Thr</td><td> Leu</td><td> Thr</td><td> Leu</td><td> Asp</td><td> Thr</td><td> Ser</td><td> Ser</td><td> Ser</td><td> Thr</td><td> Ala</td><td> Tyr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td> Met</td><td> Gin</td><td> Leu</td><td> Ser</td><td> Ser</td><td> Leu</td><td> Thr</td><td> Ser</td><td> Gxu</td><td> Asp</td><td> Ser</td><td> Ala</td><td> Val</td><td> Tyr</td><td> Tyr</td><td> Cys</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td> Ser</td><td> Arg</td><td> Gly</td><td> Asp</td><td> Gly</td><td> Ser</td><td> Asp</td><td> Tyr</td><td> Tyr</td><td> Ala</td><td> Met</td><td> Asp</td><td> Tyr</td><td> Trp</td><td> Gly</td><td> Gin</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td> Gly</td><td> Thr</td><td> Thr</td><td> Val</td><td> Thr</td><td> Val</td><td> Ser</td><td> Ser</td><td> Gly</td><td> Gly</td><td> Gly</td><td> Gly</td><td> Ser</td><td> Asp</td><td> He</td><td> Glu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td> Leu</td><td> Thr</td><td> Gin</td><td> Ser</td><td> Pro</td><td> Ala</td><td> He</td><td> Leu</td><td> Ser</td><td> Ala</td><td> Ser</td><td> Pro</td><td> Gly</td><td> Gly</td><td> Lys</td><td> Val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td> Thr</td><td> Met</td><td> Thr</td><td> Cys</td><td> Arg</td><td> Ala</td><td> Ser</td><td> Ser</td><td> Ser</td><td> Val</td><td> Ser</td><td> Tyr</td><td> Met</td><td> His</td><td> Trp</td><td> Tyr</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td> Gin</td><td> Gin</td><td> Lys</td><td> Pro</td><td> Gly</td><td> Ser</td><td> Ser</td><td> Pro</td><td> Lys</td><td> Pro</td><td> Trp</td><td> He</td><td> Tyr</td><td> Ala</td><td> Thr</td><td> Ser</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td> Asn</td><td> Leu</td><td> Ala</td><td> Ser</td><td> Gly</td><td> Val</td><td> Pro</td><td> Thr</td><td> Arg</td><td> Phe</td><td> Ser</td><td> Gly</td><td> Thr</td><td> Gly</td><td> Ser</td><td> Gly</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td> Thr</td><td> Ser</td><td> Tyr</td><td> Ser</td><td> Leu</td><td> Thr</td><td> lie</td><td> Ser</td><td> Arg</td><td> Val</td><td> Glu</td><td> Ala</td><td> Glu</td><td> Asp</td><td> Ala</td><td> Ala</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td> Thr</td><td> Tyr</td><td> Tyr</td><td> Cys</td><td> Gin</td><td> Gin</td><td> Trp</td><td> Ser</td><td> Arg</td><td> Asn</td><td> Pro</td><td> Phe</td><td> Thr</td><td> Phe</td><td> Gly</td><td> Ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td> Gly</td><td> Thr</td><td>^Lys</td><td> Leu</td><td> Glu</td><td> He</td><td> Lys</td><td> Arg</td><td> Ala</td><td> Ala</td><td> Ala</td><td> Glu</td><td> Gin</td><td> Lys</td><td> Leu</td><td> He</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td> Ser</td><td> Glu</td><td> Glu</td><td> Asp</td><td> Leu</td><td> Asn</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

245 (2) INFORMATION FOR SEQ ID NO: 58:

<td> (i)</td><td colspan="8"> SEQUENCE CHARACTERISTICS: (A) LENGTH: 101 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear</td>

<td> (ii)</td><td> MOLECULE TYPE: protein</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> (xi)</td><td> SEQUENCE DESCRIPTION; SEQ IE</td><td> i NO: 58 :</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Met 1</td><td> Ser Asn Thr Gin Ala Glu Arg 5</td><td> Ser He 10</td><td> He</td><td> Gly</td><td> Met</td><td> He</td><td> Asp 15</td><td> Met</td>

<td> Phe</td><td> His Lys Tyr Thr Arg Arg Asp 20</td><td> Asp Lys 25</td><td> He</td><td> Asp</td><td> Lys</td><td> Pro 30</td><td> Ser</td><td> Leu</td>

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154

<td> Leu</td><td> Thr</td><td> Met 35</td><td> Met</td><td> Lys</td><td> Glu</td><td> Asn</td><td> Phe 40</td><td> Pro</td><td> Asn</td><td> Phe</td><td> Leu</td><td> Ser 45</td><td> Ala</td><td> Cys</td><td> Asp</td>

<td> Lys</td><td> Lys 50</td><td> Gly</td><td> Thr</td><td> Asn</td><td> Tyr</td><td> Leu 55</td><td> Ala</td><td> Asp</td><td> Val</td><td> Phe</td><td> Glu 60</td><td> Lys</td><td> Lys</td><td> Asp</td><td> Lys</td>

<td> Asn 65</td><td> Glu</td><td> Abp</td><td> Lys</td><td> Lys</td><td> lie 70</td><td> Asp</td><td> Phe</td><td> Ser</td><td> Glu</td><td> Phe 75</td><td> Leu</td><td> Ser</td><td> Leu</td><td> Leu</td><td> Gly 80</td>

<td> Asp</td><td> lie</td><td> Ala</td><td> Thr</td><td> Asp</td><td> Tyr</td><td> His</td><td> Lys</td><td> Gin</td><td> Ser</td><td> His</td><td> Gly</td><td> Ala</td><td> Ala</td><td> Pro</td><td> Cys</td>

90 95

Ser Gly Gly Ser Gin 100