Improved method for the refolding of proteins

Field of Invention The present invention relates to recombinant DNA technology, in particular, efficiently in vitro misfolded and / or insoluble proteins containing proteins containing disulfide bonds. In vitro) Protein engineering to produce properly folded proteins by expression of genes or gene fragments in host organisms that are heterologous or homologous as recombinant protein products by describing new general principles and methodologies for regeneration. Regarding technology. The present invention further relates to the regeneration of unfolded or misfolded polypeptides of any other origin. The present invention also relates to a novel design of a site that is an encrypted recognition site for factor Xa cleavage of a chimeric protein and is only recognized after in vitro derivatization. We also provide two analogs of bovine coagulation factor Xa. This analog is suitable for small, medium or large scale technical applications involving specific cleavage of chimeric proteins at sites designed for cleavage by factor Xa. Finally, the present invention relates to the design of reversible disulfide blocking reagents. This reagent is useful as an adjunct compound for the regeneration of cysteine-containing proteins, including general analytical procedures that can assess the suitability of disulfide exchange reagents for this particular purpose. General Background of the Invention The technique of producing virtually any polypeptide by introducing a natural or synthetic DNA fragment encoding a particular polypeptide into a suitable host by recombinant DNA methods has been available for more than 15 years. Developed enthusiastically, it is now the primary tool for many industrial processes to produce high grade protein products for biochemical research and for biomedical and other industrial applications. It has become. The four basic physical characteristics of the biological system make it possible to produce proteins heterogeneously: (i) The functional properties of a protein are fully identified by its tertiary structure and, due to the molecular environment in this structure, are expressed by the chemical properties exhibited by a particular part of this structure. (ii) The tertiary structure of the protein is then identified by sequence information represented by a particular contiguous sequence of amino acid residues in a straight peptide chain. The structural information embedded in the amino acid sequence of the polypeptide is itself sufficient under conditions suitable for performing the folding process, where the final product is a complete and properly folded protein. (iii) The linear sequence of amino acid residues in a polypeptide chain is identified by the nucleotide sequence in the coding region of the genetic material that assembles the polypeptide chain by cellular mechanisms. Translation tables that determine the translation of nucleic acid sequence information into amino acid sequences are known and are almost common in known organisms. Thus, the translation table allows nucleic acid segments encoding any polypeptide segment to assemble peptide products across virtually any heterologous barrier. (iv) Individual types of organisms depend on the characteristic sequences of genetic elements present in the gene, in response to specific intracellular and extracellular factors, of a given gene with respect to transcription and translation. It interacts with the cellular molecular mechanisms that regulate expression. Therefore, in order to substantially produce the desired recombinant protein product by utilizing the protein synthesis mechanism of the host cell or organism, the DNA segment encoding the desired product is recognized by the cell's genetic control system. It needs to be presented to the fused cells to control the sequence. Immediate fate of the polypeptide expressed in the host fate) is affected by the nature of the polypeptide, the nature of the host, and the stress state of the host organism that can be evoked during the production of a given polypeptide. Gene products that are expressed at moderate levels and that are similar or identical to proteins normally present in host cells are normally processed and accumulated or secreted in the appropriate cell compartment, regardless of the natural fate of this endogenous gene product. Often experienced. In contrast, recombinant gene products that are foreign or produced at high levels to cells have a cell defense mechanism that resembles a cell defense mechanism that is activated by exposure to heat shock or toxic amino acid analogs (ie,). Originally designed to allow cells to escape from "wrong" polypeptide material by controlled intracellular proteolysis or segregation of unwanted polypeptide material into storage particles ("encapsulation"). Often activates the pathways that exist. Recombinant proteins in these stored particles often precipitate in an accidentally folded or aggregated state, in which case the product is dissolved under denaturing and reducing conditions to obtain a useful protein product. Then, it is necessary to fold the recombinant polypeptide by an in vitro method. Expression of eukaryotic genes in eukaryotic cells allows the properly folded and processed gene product to be isolated from cell culture medium or cell material. This approach was often used to obtain relatively small amounts of protein for biochemical research and is now also industrially utilized to produce numerous biomedical products. However, eukaryotic expression technology is expensive in terms of technical complexity, labor costs, and material costs. Moreover, in terms of the developmental time required to establish an expression system, laboratory-level production alone can take at least several months. The nature and degree of post-translational modification of recombinant products often differs from the nature and degree of natural products. This modification is indirectly genetically regulated in the host cell. Sequence signals that trigger post-synthesis modification are often among eukaryotes. Although often mutually recognized, the effectiveness of the appropriate site of the modifying enzyme is determined by the nature and condition of the host cell. Various strategies have been developed for the expression of gene products in prokaryotic hosts, which are more advantageous than eukaryotic hosts in terms of financial, labor and material requirements. Strains of Escherichia coli, which are eubacteria, are often preferred as host cells. This is because E. coli is genetically much more characterized at the molecular level than any other organism. Prokaryotic host cells do not have the enzymatic mechanisms required to make post-translational modifications, so eukaryotic gene products will always be produced in their unmodified form. In addition, the product results from the N-terminal extension, the required translation initiation codon, which more often contains the N-terminal segment corresponding to the N-terminal segment of the highly expressed host protein. It must be synthesized with at least one additional methionine residue. Also described are common methods for removing such N-terminal proteolysis by sequence-specific proteolysis at the linker segment inserted at the junction between the N-terminal extension and the desired polypeptide product. {Linker sequence cleavable by enterokinase: EP 035384, The Regents of the University of California; Linker sequence cleavable by factor Xa: EP 161937, nagai &amp; amp Thogersen, Transferee: Celltech Ltd. }. Strategies for heterologous expression in prokaryotes to generate soluble forms of recombinant protein products, or fusion protein constructs that are secreted from cells in an active, possibly N-terminally treated form. Considerable efforts have been made towards development over the years, but this effort has had limited success despite recent developments in the chaperone field. Typically, much time and effort is required to develop and modify the expression system before isolating a small amount of soluble and properly folded fusion protein product. More often, all of the polypeptide products are precipitated in the host cell in an improperly folded state in the "encapsulation". This is especially true for the expression of eukaryotic proteins containing disulfide bridges. All effective methods for in vitro regeneration of proteins are to expose the solvent to a protein that is present in solution or that is non-specifically adsorbed on an ion exchange resin or the like, and the composition of the solvent is made at once ( single pass) over time time) A step of gradually changing from strong denaturation (and sometimes reduction) to non-denaturation is described. This involves diluting a concentrated solution of protein containing 6-8 M of guanidine hydrochloride or urea into a substantial amount of non-denaturing buffer, or dialyzing a diluted solution of protein in the modified buffer against the non-denaturing buffer. Often done by things. Many variations of this basic procedure have been described, including those that add specific ligands or cofactors for active proteins that appear to stabilize the folding structure, such as polyethylene oxide (polyethylene glycol). Includes those that add a polymeric substance. Effective variants of the standard procedure for in vitro regeneration have been found for many specific protein products, including proteins containing one or more disulfide bonds, but regeneration yields are often lower and incomplete. Increasing yields is impractical and costly due to the low solubility of most proteins folded into and the need for excessive solvent usage. A common feature of all traditional in vitro regeneration protocols is that regeneration, which is induced by a rapid or gradual decrease in denaturation, is performed as a single operation and its yield is the protein in question. It is considered to be the largest available. The general field of protein folding is outlined in a recent instructional book by Thomas W. Creighton ("Protein Folding", Creighton TE, Freeman 1992), which provides practical protein regeneration. A more specific study of the method was published in 1989 by Rainer Jaenicke &amp; Rainer Rudolph ("Protein Structure, Practical Approach", edited by TE Creighton, IRL Press 1989). Of the many publications described in more detail, by Schein (Schein CH, 1990, You may refer to the one which examined the technical state such as 157-162). Therefore, there is a need for generally applicable high yield methods for regenerating unfolded or misfolded proteins from various sources such as prokaryotic expression systems or peptide synthesis. It's clear. Outline of the Invention It has been found by the present inventors that the yield of regeneration can be greatly increased by considering the following. That is, the protein folding process is a dynamically controlled process, and between the folded and unfolded conformers of the protein and the misfolded conformers. Mutual conversion accumulates in a cyclical denaturation-restoration step (regenerated protein products increase at each cycle at the expense of unfolded and misfolded conformers). ) Can be utilized to generate new regeneration processes with much greater potential than the basic traditional approach, by being susceptible to denaturation and time-dependent phenomena. is there. The term "folded protein" is a polypeptide in a conformation that corresponds to the conformation that occurs in a biologically active form of protein, or a unique stable intermediate (converted in subsequent steps). It can also give rise to biologically active species). The covalent structure of a folded protein in terms of cross-linking between paired cysteine residues in a polypeptide is identical to the covalent structure of a biologically active form of the protein. Therefore, the term "unfolded protein" is in a conformational state that is less compact and less clearly defined than the polypeptide that is biologically active and therefore corresponds to the folded form of the protein. Refers to a polypeptide. The covalent structure of an unfolded protein in terms of cross-linking between paired cysteine residues in a polypeptide may be the same or not identical to the covalent structure of a biologically active form of the protein. Unfolded ta Closely related to the impact is a thermodynamically stable conformation, sometimes even more stable than the conformation corresponding to the folded form of the protein. It is a "misfolded protein" that is a polypeptide that is in a conformed state and does not exhibit bioactivity to the same extent as a folded protein, even if it exhibits biological activity. As in the case of unfolded proteins, the covalent structure in terms of cross-linking between paired cysteine residues in the polypeptide may be the same or not identical to the covalent structure of the folded protein. The term "regenerated protein" is a covalent structure in terms of a transformed from the unfolded state and a bioactive conformation and cross-linking between the correctly paired cysteine residues in the polypeptide. Means a polypeptide that takes. This newly applicable strategy for protein regeneration is designed on the basis of the following general properties of protein structure: (a) The low solubility of unfolded proteins exposed to a non-denaturing solvent induces the polypeptide to form a compact, correctly regenerated structure, or is misfolded within a reasonable amount of time. It reflects the major driving force that produces stagnation aggregates or precipitates that cannot be regenerated under non-denaturing conditions to produce properly regenerated structures. (b) Newly formed stagnation aggregates are more easily "denatured", i.e. transformed into unfolded forms, than properly regenerated proteins. This is due to the more disordered structure of the Yukidomari agglomerates. Perhaps incorrect folding is also a generally dynamically controlled process. (c) Unfolded proteins are often unable (or very slow) to regenerate into the properly regenerated form at the denaturing level required to denaturize and denature aggregates within a reasonable amount of time. Only possible with). (d) A set of evidence available to support (b) includes detailed studies of folded and unfolded pathways and intermediates for several model proteins. Also, the stability of disulfide bonds to reductions and reductions at limiting concentrations of denaturants is often significantly different for each disulfide bridge of a given protein, and the disulfide bridges of the folded protein are "non-denatured" in the denatured protein or protein aggregate. Observations have also been made for many disulfide-bonded proteins that are generally much less susceptible to reduction or disulfide exchange than non-native "disulfide bonds. The new strategy for the regeneration procedure is most easily described based on the following theoretical example: Assumed protein-non-denaturing buffer "A" that was exposed to buffer A or buffer B and then incubated at intermediate levels of denaturation in a mixture of buffer A and buffer B, folds stably. It is assumed that it does not fold stably in the strong denaturing buffer "B" (buffer B contains, for example, 6M of guanidine hydrochloride). When buffer B is in the 100-75% range, both folded and squeezed aggregated proteins are quickly converted to unfolded form. When buffer B is in the 75-50% range, newly formed stagnation aggregates are converted to unfolded form, while almost all of the regenerated protein is within at least a few hours. It has a stable, natural-like structure and will immediately return to its regenerated form upon removal of the denaturant. When buffer B is 10% or more, rapid formation from the unfolded form to the regenerated form is inhibited. When the solvent composition step was changed from 100% buffer B to 0%, the unfolded protein was converted to a stagnation aggregate (75% yield) and a regenerated protein (25% yield). To. A sample of this protein, initially in unfolded form in 100% buffer B, was sampled in a time-series programmed denaturation-restoration cycle, as shown in Figure 1. Fn) (consisting of &lt;10% B) and denatured phase (Dn)). At the end of the restoration phase of period (i), the denatured contents change to a level ki% lower than the degeneration level of the previous period. After a brief incubation, the denaturant is removed again and the next convalescent phase Fi + 1 is entered. Assuming that the denaturation level starts at 100% buffer and the ki of each cycle is fixed at 4%, this method will result in a series of attenuated denaturation steps ending after 25 cycles. Throughout the 25 cycles as described above, the accumulation of regenerated protein proceeds as follows: In cycles 1-5, all folded proteins, not just the misfolded proteins, will unfold in each denatured phase Dn. Cycle 7 to Cycle 12: Yukidomari aggregates are converted to unfolded protein in each step, while the amount of protein that can be restored as a regenerated product is as follows for each cycle: It will accumulate in 25%, 44%, 58%, 68%, 76% and 82%. In cycles 13-25, no conversion occurs anymore. Therefore, a periodic regeneration step will produce an overall regeneration yield of 80% or higher, while a single restoration in the best conventional conditions will produce a yield of 25%. It should be understood that with and without gradual changes in each feature of the various conformational states of this hypothetical protein are shown as approximate values for brevity. Nevertheless, the basic working principle does not make a big difference when a more complex set of estimates is added to this model. Practical configurations for establishing a cyclic denaturation / restoration protein regeneration process can be prepared in various ways. The proteins in solution can be retained, for example, in ultrafiltration membrane devices or permeation devices, or in any of the phases of a suitable aqueous two-phase system. With any of these, the concentration of low molecular weight chemical solutes in the protein solution could be controlled by a suitable device. The protein can also be absorbed on the appropriate surface in contact with the liquid phase where the chemical composition can be controlled as required. Suitable surfaces include, for example, filtration equipment, hollow fiber equipment or beaded chromatography media. Absorption of proteins into the surface is by folding-compatible covalent bonds between the surface and the protein, or by appropriately induced protein recombination that exhibits a specific and denaturation-resistant affinity for the surface. Through the specific design of the affinity handle in the derivative, it can be mediated by non-specific interactions, such as those described in WO 86/05809 (Thomas Edwin Creighton). Specific implementation of the periodic denaturation / restoration protein regeneration process established to investigate the possibility of a general method is a metal affinity handle module inserted at the N-terminus of the designed factor Xa cleavage site {EP ) Was based. This commonly designed recombinant protein, absorbed in nickel-chelate agarose beads, is then fed by an arranged calibrated pump with appropriate denaturing buffer and non-denaturing, time-programmed with a computer-controlled flow velocity. The chromatographic column "regeneration reactor" perfused with a mixture with buffer can be subjected to the periodic regeneration step of the present invention. A general plan for solid-state regeneration is outlined above in a gradual manner in which the concentration of denaturant decreases from a high initial value to gradual zero throughout many series of restoration-denaturation cycles. It includes the step of circulating the immobilized protein as such, or by any other means and practice between denaturing and non-denaturing conditions. When using this approach, it is not necessary to determine exactly which limiting denaturant concentration is required to increase the folding yield while circulating a particular protein at hand. This is because this gradual sequence of cycles is (at most) three phases, that is, the folded product present at the end of cycle (i) is completely denatured in the denaturation step of cycle (i + 1). Because the periodic phase, the intermediate phase with increased accumulation of regenerated products, and the concentration of denaturant passes through the final phase, which is too low to perturb the regenerated protein or any remaining misfolded structure. Is. For disulfide-containing proteins, increase the cycle of gradual denaturation-restoration using a device similar to an improved chromatographic device equipped with online equipment to monitor the buffer composition of the effluent effluent of the folding reactor. Can be done. Information about the effluent composition of the reducing agent and the concentration profile of the disulfide reshuffling reagent reveals the production cycle, so this information can be used for input to processing equipment with information processing capabilities, and then the cycle. The gradual progression of the denaturant concentration in the feedback loop can be adjusted to ensure that most of the force is consumed within the production phase of the series of denaturation / restoration cycles. Cycle line Such automatic optimization of the case is possible because the analytical system can be used to measure the degree and direction of changes in the redox equilibrium of the buffer stream, and this measurement is the thiol in the immobilized protein sample. This is because it directly reflects the titration of group / disulfide equivalents and can therefore be immediately translated into the average number of disulfide bonds broken or formed in the various phases of the cycle. Other possible inputs to processing equipment with information processing capabilities that control the progression of the cycle are ligand binding, substrate conversion, antibody binding capacity, and proteins that are misfolded and folded in a well-defined manner ( Any other solubilizable that interacts with the effluent (which can also be percolate through a regenerative reactor during the rating phase of the fold measurement and then monitored in-line in the effluent with a suitable analyzer). Measurement of the interacting agent can be mentioned. Information processing monitoring and control systems also use available information to salvage / recycle the available portion of the reactor effluent to the salvage / recycling subsystem, thereby minimizing the cost of large-scale operations. can do. After performing the folding step, the final product is eluted from the affinity matrix in concentrated form and processed to release mature euprotein by cleavage at the designed protease cleavage site, followed by standards known in the field of protein chemistry. Protein purification and processing techniques allow for final post-treatment. Detailed Disclosures of the Invention Therefore, the present invention comprises at least one modification step in which an initial assembly of polypeptide molecules (ensemble) is subjected to a series of 1) conditions that have a modifying effect on the polypeptide molecule of the assembly. Subsequent 2) Poly consisting of at least two consecutive period sequences consisting of at least one restoration step with conditions that have a modifying effect on the conformationally constitutive polypeptide molecule obtained from the above steps. From the initial aggregate of polypeptide molecules having the same amino acid sequence as the treated aggregate of peptide molecules, the treated aggregate The present invention relates to a method for generating a processed aggregate of a polypeptide molecule containing a substantial fraction of the polypeptide molecule in a particular uniform conformation with the conformational state represented in. In the specification and claims, the term "ensemble" is used in the sense that the term has acquired in the art. That is, the term refers to a collection of molecules with major common characteristics. Initially ("early aggregates"), they share at least their amino acid sequence in common (and, of course, retain this common feature). When an aggregate of polypeptide molecules is processed by the method of the invention (to obtain a "processed aggregate"), the conformational state represented in this aggregate has one particular conformation. Will contain a substantial fraction of the polypeptide molecule. As will be understood from the following description, the substantial fraction of a polypeptide molecule having one particular conformation in a treated aggregate is a parameter of the treatment according to the method of the invention, a protein in a particular conformation. Will vary depending on the size of the molecule, the length and identity of the amino acid sequence of the molecule, and so on. In the examples where the processing parameters reported here have not yet been optimized, the fraction of the polypeptide molecule with one particular conformation is 15% to 100% of the aggregate (in each case). , Which is higher than the numbers that could be obtained before this invention). Example 13 further shows that purification of the polypeptide molecule prior to the method of the invention increases the fraction of the polypeptide molecule with one particular conformation. The "denaturing step" refers to an aggregate of polypeptide molecules at time intervals, and an aggregate of polypeptide molecules under conditions characterized by a more intense denaturing power than the denaturing power that characterizes the condition immediately prior to the denaturing step. Refers to exposure to the physical and / or chemical environment attached. Therefore, the term "renaturing" "Step)" means that an aggregate of polypeptide molecules is attached at time intervals to a condition characterized by a denaturing force that is less severe than the denaturing force that characterizes the condition immediately before the denaturation step. And / or refers to exposure to the chemical environment. The magnitude of the "substantial fraction" described above will depend on the assembly of polypeptide molecules attached to the method of the invention. When the treated aggregate of a polypeptide is composed of a monomeric protein that is relatively short in length and does not have an intramolecular disulfide bridge, the methods of the invention generally yield very high yields. As will be obtained, complex molecules (such as multimeric proteins with complex disulfide cross-linking topologies) will yield low yields, even if the conditions of the methods of the invention are fully optimized. Let's go. An interesting aspect of the invention is that the treated aggregate is a substantial fraction of the polypeptide molecule in one conformational state, which constitutes at least 1% (W / W) of the initial aggregate of the polypeptide molecule. It relates to the above method consisting of fractions. Higher yields, such as at least 5%, at least 10%, at least 20% and at least 25% of the initial assembly of polypeptide molecules are preferred. More preferred yields are at least 30%, such as at least 40%, 50%, 60%, 70% and at least 80%. Particularly preferred yields are at least 85%, such as 90%, 95%, 97%, and 99%. Sometimes yields close to 100% are observed. If the polypeptide molecule of the aggregate contains cysteine, the treated aggregate is disulphide bridging, which is substantially identical. Consists of a substantial fraction of the polypeptide molecule in one particular uniform conformation with a further topology). In most cases, the polypeptide molecule attached to the method of the invention is a molecule having the same amino acid sequence as the amino acid sequence of a genuine polypeptide, or is attached to one or two additional polypeptide segments. It will be a molecule consisting of an amino acid sequence corresponding to the amino acid sequence of a genuine polypeptide. The term "genuine protein or polypeptide" means a polypeptide having a primary structure containing N-terminal and C-terminal structures that is identical to the primary structure of the corresponding native protein. The term also refers to a polypeptide having a known primary structure that is not necessarily identical to the primary structure of a natural protein and is the intended end product in protein synthesis. The term "natural protein" means a protein isolated in a biologically active form from an organism in which the protein is present rather than as a result of genetic engineering. In contrast, the term "artificial protein or polypeptide" used in this specification and claims is not available from any natural source, i.e. isolated and purified from any natural source. Intended for proteins / polypeptides that cannot. Thus, artificial proteins / polypeptides can be the result of human intervention, eg, the product of recombinant DNA manipulation or a form of in vitro peptide synthesis. According to the above definition, these artificial proteins may be genuine proteins and not natural proteins. Thus, the present invention relates not only to artificial proteins but also to methods of subjecting natural proteins to the regeneration steps described herein. As described in more detail below, expression of an authentic polypeptide, for example, as a "handle" for binding the polypeptide to a carrier, as a solubility modifier, exercising useful functions during translation of messenger RNA, etc. As boosters, a polypeptide segment that has ancillary functions during circulation and other treatments before and after it. It is advantageous for various reasons to be bound to. Such an auxiliary polypeptide segment is preferably attached to the authentic polypeptide via a cleavable bond, and when two such ancillary polypeptide segments are attached to the authentic polypeptide, this binding is usually It can be made through similar cleavable bonds that are cleaved at the same time or dissimilar cleavable bonds that can be cleaved in any time series. According to the above, the main novel feature of the present invention is that the denaturation step (at least the substantial fraction of the regenerated polypeptide is re-denatured) after the restoration step by the circulation consisting of at least two consecutive cycles as described above. It is believed that at least one event followed by) will occur. In most cases, the processing will consist of at least 3 cycles, often at least 5 cycles and more often at least 8 cycles, eg at least 10 cycles, and in some cases at least 25 cycles. On the other hand, a series of cycles will usually not exceed 2000 cycles and will often consist of up to 1000 cycles and more often up to 500 cycles. The number of cycles used will depend in part on the potential provided by the device on which the circulation takes place. Therefore, when the circulation treatment is performed using a polypeptide molecule immobilized on a carrier column, as described in more detail below, the rate at which the liquid phase in contact with the column can be exchanged is increased. It will set one limit on what can be achieved in reality. On the other hand, high performance liquid chromatography (HPCL) equipment will change the liquid environment very quickly, thus making cycles in the range of hundreds or thousands realistic. Other possible factors that determine the desired number of cycles are, for example, proline residues that tend to complicate redistribution to partially folded states and therefore require careful timing. Inherent kinetic parameters such as interconversion between cis and trans isomers in. Another time-critical A feature lies in the dynamics of disulphide reshuffling (see the discussion below on disulfide-reorganization systems). With due consideration of the above, the circular sequence will often consist of up to 200 cycles, more often up to 100 cycles and even more often up to 50 cycles. According to the above, the duration of each modification step will be at least 1 millisecond up to 1 hour under certain conditions of concern. The duration of the restoration process will be at least 1 second up to 12 hours under certain conditions of concern. In most specific examples of this method, the modification conditions in each individual modification step are kept substantially constant for a certain period of time, and the restoration conditions in each individual restoration step are kept substantially constant for a certain period of time. Those times during which are kept substantially constant are separated by a transition period in which the conditions change. The transition time between processes in which the conditions are kept substantially constant is, for example, 0. It can have a wide range of varying durations, such as 1 second to 12 hours, and will usually fit perfectly closely with the duration of the denaturation and restoration steps. With this in mind, the time for which the modification conditions in the modification step are kept substantially constant can have a duration of, for example, at least 1 millisecond to a maximum of 1 hour, often up to 30 minutes, and in the restoration step. The time that the restoration conditions remain substantially constant has a duration of at least 1 second to a maximum of 12 hours, and often a maximum of 2 hours. In fact, the time that the modification conditions in the modification step are kept substantially constant often has a duration of 1 to 10 minutes, and the time that the restoration conditions in the restoration step are kept substantially constant often. It will have a duration of 1-45 minutes. It will be understood from the above that the above intervals need to be adjusted to account for the changes in kinetics due to the changes in the physical conditions to which the polypeptide is attached. For example, when using an HPLC system in performing the methods of the present invention, the pressure is very high (up to 5000 bar) and under these circumstances very rapid steps will be performed and / or will be required. Furthermore, as understood from the examples, temperature parameters are important because some proteins are appropriately regenerated at temperatures outside the physiological range. The time intervals of the restoration and modification steps are realistic boundaries for many possible embodiments of the invention, as both temperature and pressure naturally affect the dynamics of the regeneration process of the invention. One of ordinary skill in the art will be able to determine appropriate conditions for a given use of the methods of the invention, such as on the basis of preliminary experiments. As described above, the polypeptide molecule is in normal contact with the liquid phase during the denaturation and restoration steps, which is usually the aqueous phase. That is, any reagent or auxiliary substance used in the method of the present invention will usually dissolve in a liquid phase, usually an aqueous phase. However, if convenient, the liquid phase may be composed of one or more organic solvents. For protein restoration, the so-called "chaperone" Is known to use a "chaperone complex". Chaperones are a group of recently described proteins that exhibit common features in their ability to enhance the regeneration of unfolded or partially folded proteins. Often, chaperones are multimolecular complexes. Most of these chaperones are heat shock proteins, which are in vivo (in vivo) as a factor that "repairs" proteins that have lost stability due to trauma. It means a protein that acts in vivo). To perform this function, chaperones tend to be more stable to traumatic events than many other proteins and protein complexes. The methods of the invention do not rely on the use of molecular chaperones or molecular chaperone complexes, but it is of course possible and substantially possible to have suitable molecular chaperones or molecular chaperone complexes present during at least one restoration step. It is preferable to have a molecular chaperone or a molecular chaperone complex that is present during the entire cycle. As mentioned above, it is preferred that the polypeptide molecule be substantially confined in an environment in which the liquid phase can be altered or exchanged without substantially entraining the polypeptide molecule. This can be achieved in many ways. For example, the polypeptide molecule can be contained in a dialysis machine or confined in one phase of a suitable liquid two-phase system. Such a suitable aqueous two-phase system can contain, for example, a polymer selected from the group consisting of polyethylene oxide (polyethylene glycol), polyvinyl acetate, dextran and dextran sulfate. In one interesting configuration, one phase will contain polyethylene oxide (polyethylene glycol) and the other phase will contain dextran, whereby the polypeptide molecule will be trapped in the dextran-containing phase. Another way to avoid floating polypeptides is to use polypeptide molecules, such as filter surfaces, hollow fiber or beaded chromatographic media (agarose, polyacryamide gel, etc.), fibrous cellulose matrix, etc. Or HPLC or FPLC {Fast Performance Liquid Chromatography)} To bind to a solid or semi-solid carrier such as a matrix. Alternatively, the carrier is a substance having a size that allows the molecule to which the polypeptide molecule is bound to be retained by the filter when dissolved or dispersed in the liquid phase, or the carrier is a micelle. It may be a substance capable of forming micelles or participating in the formation of micelles while changing or exchanging the liquid phase without substantially floating and carrying. If the micelle-forming component attempts to escape the system as a monomer (for example, if it is possible to pass through the ultrafiltration membrane used to limit this system to some extent), this will result in additional micelle formation. It can be compensated by replenishing the monomer. The carrier may be a water-soluble polymer having molecules of a size that is substantially impenetrable to the pores of the filter or other means used to confine the system. It is appropriate that the polypeptide molecule is non-covalently absorbed into the carrier via a component that has an affinity for the component of the carrier. Such a component may be, for example, a biotin group attached to the amino acid portion of the polypeptide or an analog thereof, and has a strong affinity between the polypeptide molecule thus modified and the carrier thus modified. To establish the system, the carrier has avidin, streptavidin or their analogs bound to them. The affinity between the modified polypeptide and the modified carrier should be sufficiently stable that adsorption is substantially unaffected by the modification conditions and the polypeptide molecule from the carrier after circulation. It will be appreciated that the removal of is required to be done by specific cleavage as described below. An example of a suitable amino acid residue to which a biotinyl group binds is lysine. One interesting method of introducing amino acids that carry moieties that have an affinity for the carrier is CPY synthesis. CPY Luboxy peptidase Y) is known to be able to add an amino acid amide regardless of the side chain properties of the amino acid amide. In one interesting embodiment, the moiety that has an affinity for the carrier is polypeptide segment SEQ ID NO: 47, in which case the carrier is Ni⁺⁺Ion-charged nitrilotriacetic acid derivative (NTA), eg Ni⁺⁺It is appropriate to consist of an NTA-agarose matrix bathed in a solution consisting of. An important aspect of the invention relates to the existence of suitable means in a polypeptide molecule that creates a molecule that is subsequently cleaved into two or more segments, one segment being the authentic polypeptide as defined above. Such combined polypeptide molecule (fused polypeptide molecule) may consist of a polypeptide segment capable of preferential cleavage with a cleavage agent at a specific peptide bond for this purpose. The polypeptide segment in question may be a polypeptide segment that cleaves as a result of the conformation of the segment that is the recognition site for the cleaving agent. Clavage-directing polypeptide The segment) could be preferentially cleaved with a specific peptide bond by a cleavage agent selected from the group consisting, for example, bromocyan, hydroxylamine, iodosobenzoic acid and N-bromosuccinimide. The polypeptide segment to be cleaved may be, for example, a segment capable of preferential cleaving with a specific peptide bond by a cleaving agent such as an enzyme, and one such possible enzyme is bovine enteropeptidase or its analogs. Examples include bodies and / or homologues. According to an important aspect of the invention, the cleaving agent is the enzyme bovine coagulation factor Xa or its analogs and / or homologues (analogs are described in more detail below) and perform preferential cleaving. The polypeptide segment is a sequence that is substantially selectively recognized by the enzyme bovine coagulation factor Xa or its analogs and / or homologues. An important such segment is a polypeptide segment having 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 ability to cleave at specific peptide bonds, thereby allowing different segments of the polypeptide to cleave at different stages of the cycle, masking and unmasking the polypeptide segments. It is possible to do. Thus, if the polypeptide molecule consists of a polypeptide segment that can be converted in vitro into an induced polypeptide segment that can be preferentially cleaved with a cleavage agent at a specific peptide bond, the masking / unmasking effect described above. Can be obtained. In a particularly interesting modification of this strategy, the in vitro convertible polypeptide segment becomes an induced polypeptide segment that is substantially selectively recognized by bovine coagulation factor Xa or its analogs and / or homologues. It is convertible. Both cysteine and methionine residues are transformed in vitro into segments recognized by the enzyme bovine coagulation factor Xa or its analogs and / or homologues. It is believed to be converted to modified residues that make up a segment with an amino acid sequence selected from the group consisting of convertible SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46. According to the present invention, in one possible solution involving a cysteine residue, a polypeptide segment having the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 44 has a cysteine residue of N- (2-mercaptoethyl) morpholyl-2. By reacting with -thiopyridyl disulfide or mercaptothioacetate-2-thiopyridyl-disulfide, it is converted into an inducible polypeptide that is substantially selectively recognized by bovine coagulation factor Xa. In a possible strategy according to this invention with methionine, SEQ ID NO:: The polypeptide segment having the amino acid sequences of 45 and SEQ ID NO: 46 is induced to be substantially selectively recognized by bovine coagulation factor Xa by oxidizing the thioether moiety of the methionine side group to a sulfoxide or sulfone derivative. Is converted to a polypeptide. In a preferred embodiment of the method of the invention, a cleaving segment having the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, or SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: A masked cleavage segment with 46 amino acid sequences is attached to the authentic polypeptide at the N-terminal. This is because no further treatment other than selective cleavage is required to obtain a genuine polypeptide in solution. On the other hand, one possible reason for binding a cleaving sequence to the C-terminus of a genuine polypeptide is that the correct folding of the polypeptide molecule depends on the free N-terminus of the polypeptide molecule. Is. In such cases, some of the cleavage-directing sequences remaining after cleavage can be removed by proper use of carboxypeptidase A and carboxypeptidase B. Changes in conditions during the transition time between these steps can be achieved, according to the invention, by changing the chemical composition of the liquid phase in contact with the polypeptide molecule. Thus, denaturing a polypeptide molecule is a denaturing of the polypeptide molecule with at least one denaturing effect. The restoration of the polypeptide molecule can be achieved by contacting the liquid phase in which the compound) is dissolved, and the restoration of the polypeptide molecule is carried out for each conformational state in which the contact with the liquid phase is due to the previous step. Poly in a liquid phase containing at least one denaturing compound that is dissolved at a concentration that tends to regenerate rather than denaturing the aggregate, or substantially no denaturing compound. This is achieved by contacting the peptide molecules. The expression "compound with denaturing effect" is the folding of a polypeptide molecule that, when present as one of the solutes in the liquid phase consisting of the polypeptide molecule, leads to the unfolding of the partial or incomplete folding of the polypeptide chain. Refers to a compound that destabilizes the state. The denaturing effect of a denaturing compound increases as the concentration of the denaturing compound in the solution increases, but in addition, due to the presence of other solutes in the solution or physical parameters such as temperature or pressure. May be enhanced or mitigated by changes in. Examples of suitable denaturing compounds used in the method of the invention are urea, guanidine hydrochloride, di-C._1-6Alkylformamide (dimethylformamide, di-C_1-6(Alkyl sulfone, etc.). The liquid phase used in at least one of the modification steps and / or at least one of the restoration steps contains at least one disulfide-reorganization system according to the invention. A "disulfide-reorganization system" is a redox system containing a mixture of a reducing agent and an oxidant, and the presence of this mixture of the reducing agent and the oxidant disrupts the disulfide bond in or between the polypeptides. And formation is promoted. Thus, a "disulfide reorganization agent" or "disulfide reorganization compound" is a reducing agent and oxidizing agent that promotes the disruption and formation of disulfide bonds in or between polypeptides. In an important aspect of the invention, the disulfide reorganization system contained in the aqueous phase in contact with the protein contains, as the disulfide reorganization system, a mixture of mercaptan and the corresponding disulfide compound. As an example, all cysteine residues in a polypeptide molecule are converted to a mixed disulfide product of either glutathione, thiocholine, mercaptoethanol or mercaptoacetic acid during at least one denaturation / restoration cycle. Such converted polypeptides are named "disulfide-completely blocking polypeptides or proteins", hence the term cysteine residues are disulfide-bonded to mercaptans such as glutathione. Refers to a polypeptide or protein converted into a mixed disulfide. Conversion of cysteine residues to mixed disulfide products results in complete modification of polypeptide molecules and high energy mixed disulfides such as excess aliphatic-aromatic disulfide compounds (such as 2-thiopyridylglutathionyl disulfides). It can be achieved by reacting with a reagent that is a compound, or by other suitable methods. An example of a mixed disulfide with a high energy mixed disulfide, i.e. a relatively unstable SS bond), is the general formula:<img file="JP3695467B2_D0001.tif" />[In the formula, R₁Represents 2-pyridyl, R₂, R₃And R₄Represents hydrogen or optionally substituted lower aromatic or aliphatic hydrocarbon groups, respectively]. Examples of such mixed disulfides include glutathionyl-2-thiopyridyl disulfide, 2-thiocholyl-2-thiopyridyl disulfide, 2-mercaptoethanol-2-thiopyridyl disulfide and mercaptoacetate-2-thiopyridyl disulfide. In an interesting embodiment, disulfide reorganization systems include glutathione, 2-mercaptoethanol or thiocholine, each used in admixture with its corresponding symmetric disulfide. The solubility of a given mixture of thiols used as a selective reduction and / or disulfide rearrangement system in a periodic regeneration / reoxidation step for a specific protein product is weak, moderate or strong to the protein. Direct analysis by incubating a sample assembly of a mixture of folded and misfolded proteins with a series of thiol mixtures with varying concentrations of denaturant affected by be able to. After incubation, the disulfide topology in each sample is reacted with excess thiol blocking reagent (such as iodoacetamide) to lock and each set of samples is subjected to SDS-PAGE under reducing conditions. Precisely disulfide-crosslinked materials and materials with undesired covalent topological states will appear in separate bands, which will allow quantitative assessment of protein folding during thiol blockade. This is because only the correctly and unique disulfide-bonded phase isomers correspond to the correctly folded proteins present at the end of the incubation with the thiol / disulfide and the denaturant. This set of fruits is shown by the preferential reduction and rearrangement of false disulfide bonds and the low tendency to reduce bonds in fully folded proteins. Testing makes it possible to identify a range of denaturation levels at which a given thiol / disulfide reagent can be advantageously used as a disulfide reorganizer. This reagent testing step can be used as a general procedure for selecting advantageous reducing and / or thiol / disulfide reorganization reagents. Example 12 demonstrates the application of this analytical procedure to assessing suitability for selective reduction of misfolded forms in model proteins for five thiol reagents, thereby providing feasibility of the above procedure. It is shown. It will be appreciated that the above procedure for selecting a suitable disulfide reorganization system can also be used in selecting components other than mixtures of thiols. Any mixture containing a suitable reducing / oxidizing agent can be evaluated by the above procedure and the components selected in the methods of the invention have the greatest ability to preferentially reduce inaccurately formed disulfide bridges. Will show high. Thus, a very important aspect of the invention is the method for protein regeneration described herein, in which at least one contained in the liquid phase in at least one restoration and / or denaturation step. The disulfide reorganization system provides conditions for the concentration of denaturants that are substantially free of reduction and / or reorganization of unfolded and / or misfolded proteins that are denatured and accurately formed disulfide bridges. Is a system capable of reducing and / or rearranging inaccurately formed disulfide bridges. An interesting embodiment of the invention is the method described above, in which the disulfide reorganization system is used in at least one modification / restoration step and is inaccurate at least 1.05 in the early stages of reduction / reorganization. It will yield the ratio of the relative amount of disulfide bridges formed to the relative amount of disulfide bridges formed correctly in the early stages of reduction / rearrangement. This ratio is higher, for example 1.1, 1.5, 2.0, 3.0, 5. It is preferably 0, 10, 100 and 1000, but even higher ratios are practical and are particularly preferred according to the invention. The term "initially inaccurate / accurate" for the morphology of disulfide bridges is the term disulphide bridging just before the disulfide reshuuffling system is effective. means topology). It will be appreciated that the ratio must be greater than 1 in order to reticulate the correctly formed disulfide bridges in the protein sample. Generally, the ratio should be as large as possible, but even ratios barely greater than 1 can reticulate the disulfide bridges formed exactly in the method of the invention, an important parameter to ensure high yields. Is the number of degeneration / restoration cycles. If the ratio is barely greater than 1, it is necessary that many cycles have been completed before a substantial yield of accurately formed disulfide bridges can be obtained, while if the ratio is high, it is limited. Only the required number of cycles is needed. If only one disulfide reorganization system is used, such a disulfide reorganization system, according to the invention, 1) provides a series of disulfide reorganization systems in which the concentration of the selected modifier is varied in several steps. Incubates samples of folded and misfolded proteins that have the same amino acid sequence as the protein processed by the method of the invention, and 2) inaccurately formed in the early stages of reduction / rearrangement. Initial in each disulfide reorganization system, as assessed by calculating the ratio of the relative amount of disulfide bridges formed to the relative amount of disulfide bridges formed correctly in the early reduction / rearrangement. The ability to reduce / rearrange initially inaccurately formed disulfide bridges without substantially reducing / rearranging the correctly formed disulfide bridges was evaluated at different concentrations of modifiers, 3 ) Ability to reduce initially incorrectly formed disulfide bridges in the widest concentration range of selected modifiers, without substantially reducing and / or rearranging the initially accurately formed disulfide bridges. The disulfide reorganization system showing the above can be selected by selecting it as the disulfide reorganization system X. Also, one or more disulfide reorganization systems can be used, for example, in the periodic regeneration method of the present invention. Can be used simultaneously in different cycles and in the same cycle. This is because, for example, the overall yield of properly folded proteins with the correct disulfide cross-linking topology is higher than with different disulfide rearrangement systems in the methods of the invention, eg, by the methods outlined above. It may be expected or already achieved. To calculate the ratio of the relative amount of disulfide bridges that were incorrectly formed in the early reduction / rearrangement to the relative amount of disulfide bridges that were correctly formed in the early stage of reduction / rearrangement, The following methods can be used. That is, a known amount of radiolabeled, correctly folded protein is added to the initial mixture of the reactants of step 1). When assessing the amount of accurately folded protein and incorrectly folded protein in step 2) (eg, using non-reducing SDS-PAGE), the content of radioactivity in the fraction of the accurately folded protein is also decide. It allows the assessment of currently inaccurately folded (but initially correctly folded) proteins to be determined in parallel with the determination of the total distribution of accurately / inaccurately folded proteins. it can. Thus, the above ratio is When assessing the amount of folded protein in step 2) (eg, using non-reducing SDS-PAGE), the content of radioactivity in the exact folded protein fraction is also determined. It allows the assessment of currently inaccurately folded (but initially correctly folded) proteins to be determined in parallel with the determination of the total distribution of accurately / inaccurately folded proteins. it can. Thus, the above ratio is When assessing the amount of folded protein in step 2) (eg, using non-reducing SDS-PAGE), the content of radioactivity in the exact folded protein fraction is also determined. It allows the assessment of currently inaccurately folded (but initially correctly folded) proteins to be determined in parallel with the determination of the total distribution of accurately / inaccurately folded proteins. it can. Thus, the above ratio is<img file="JP3695467B2_D0002.tif" />[In the formula, C₁Is the initial amount of protein that is accurately folded, C₂Is the final amount of protein that is accurately folded, U₁Is the amount of protein that was initially incorrectly folded, A₁The radioactivity in the protein fraction that was initially accurately folded, A₂Represents the radioactivity in a protein fraction that is accurately folded late]. In addition to the above modification means, modification can also be achieved or enhanced by reducing or increasing the pH of the liquid phase. The polarity of the liquid phase used for restoration is modified according to the invention by the addition of salts, polymers and / or hydrofluoro compounds (such as trifluoroethanol). According to the present invention, denaturation and restoration of polypeptide molecules can also be achieved by direct changes in the physical parameters (temperature, pressure, etc.) to which the polypeptide molecules are exposed, or these measures are described above. It can be used to enhance or mitigate denaturation and restoration caused by other measures. However, very important practical examples of this method are denaturing solution B and renaturing. It will be understood that this is done by causing a chemical change in the liquid phase due to a change with solution) A. In this case, the concentration of one or more denaturing compounds in B is often adjusted after each cycle, and in one important example, the concentration of one or more denaturing compounds in B decays after each cycle. However, in another important embodiment, the concentration of one or more denaturing compounds in medium B is kept uniform in each cycle. A specific example of the present invention in which the concentration of the denaturing compound in medium B is kept uniform, the most productive phase of the circulating process (with respect to the precisely folded protein) was identified and accurately folded. Particularly interesting when large-scale production of proteins is desired, the preferred concentration of the denaturing compound in medium B of this embodiment is the concentration determined to ensure maximum productivity in the circulation process according to the invention. Will be understood to be. The polypeptide molecule of the assembly attached to the method of the invention usually has a length of at least 25 amino acid residues, such as at least 30 or at least 50. On the other hand, the polypeptide molecule of the aggregate usually has a length of up to 5000 amino acid residues, for example up to 2000 or 1000 or 800. As can be seen from Example 10, the method of the invention is a properly folded diabody molecule (the diabody is Holliger et al., Enabled production (described in 1993). Therefore, an important aspect of the present invention is for a polypeptide molecule consisting of an unfolded polypeptide and / or a misfolded polypeptide having the same amino acid sequence as the amino acid sequence of the monomeric fragment of the diabodies molecule. A series of 1) denaturing steps with conditions that exert a denaturing effect on the polypeptide molecules of the aggregate, followed by 2) denaturing actions on the polypeptide molecule having a conformation resulting from the above steps. Consists of a series of at least two consecutive cycles consisting of at least one restoration step with the condition of having a substantial fraction of the initial assembly of polypeptide molecules converted to a properly folded diabodies molecule fraction. It relates to a method of producing a properly folded diabody molecule, the sequence of cycles of which is adapted to be. Such a method of properly folding the diabodies is recognized in both the above scenarios and in terms of the regenerative methods of the present invention, i.e., with respect to the choice of physical / chemical conditions as well as the circulation scheme. However, an important aspect of the method of properly folding the diabodies is the method described above in which the polypeptide molecule is in contact with the liquid phase containing the disulfide reorganization system in at least one denaturation or restoration step. The preferred denaturing agent used in these liquid phases is urea, and the preferred disulfide reorganization system consists of glutathione as the primary reducing agent. A particular aspect of the invention is that unlike all naturally occurring serine proteases, which are pro-enzymes of serine proteases, in particular bovine coagulation factor X {Protein Identification Resource (PIR), National Biomedical Research. Foundation, Georgetown University, Medical Center, USA, entry: P1; Each ratio of k (I) / k (V) and k (III) / k (V) to the cleavage rate for benzoyl-Ile-Glu-Gly-Arg-paranitroanilide is substantially mixed. Is equal to or better than the substrate specificity of bovine blood coagulation factor Xa, as assessed by subtracting from or equal to the corresponding proportion quantified for the protease-free bovine coagulation factor Xa. Regarding polypeptides. The characteristics of the above novel polypeptides as serine proteases are based on the use of the usual name of the term "serine protease". As is known in the art, serine proteases are enzymes believed to have a catalytic system consisting of active site serine alongside histidine residues. Activation of the enzyme from the corresponding proenzyme causes the amino group to rearrange within the peptide structure to form a salt bridge to the aspartic acid residue that precedes the active site serine residue, thereby catalytic site properties of the serine protease. Is based on the release of novel N-terminal residues that can form. The "artificial" serine proteases defined above are of significant importance used in the methods of the invention and other methods in which it is crucial to have a cleaving means that selectively cleaves proteins, even large folded proteins. Peptide cleavage means. Similar to bovine coagulation factor Xa, the artificial serine proteases defined above in activated form can selectively recognize the polypeptide segment that cleaves SEQ ID NO: 38, but what is bovine coagulation factor Xa? In contrast, they can be made with amino acid sequences that can be easily produced using recombinant DNA technology. As described above, the preferred artificial serine protease of the present invention has an amino acid sequence that can be synthesized by recombinant DNA technology, especially in prokaryotic cells such as Escherichia coli. As will be apparent from the discussions and examples below, the artificial serine proteases of the present invention, when produced in prokaryotes, are catalytic by circulation by the methods of the present invention. Can provide an enzymatically active conformation in which the active domain is properly exposed. Quantitative studies on the selectivity of artificial serine proteases include the determination of the cleavage rate k, which is determined as the initial slope of the light absorption curve at 405 nm (maximum absorption of free para-nitroaniline) with respect to time at 20 ° C. Is done. When expressed quantitatively, the selectivity of artificial serine proteases is characterized by up to 0.06 k (I) / k (V) values and up to 0.5 k (III) / k (V) values. Should be done. The maximum k (I) / k (V) value is 0.05, the maximum k (III) / k (V) value is preferably 0.4, and the maximum k (I) / k (V) value is 0.04. , K (III) / k (V) values are most preferably 0.15 at maximum. Additional model substrates are used to make it easier to understand and characterize specificity. Therefore, the substrate specificity is substrate I-IV: I: benzoyl-Val-Gly-Arg-para at 20 ° C, pH = 8 in a buffer consisting of 50 mM Tris, 100 mM sodium chloride and 1 mM calcium chloride. For each of Nitroanilide, II: Tosyl-Gly-Pro-Lys-Paranitroanilide, III: Tosyl-Gly-Pro-Arg-Paranitroanilide, IV: (d, 1) Val-Leu-Arg-Paranitroanilide Ratios between the cleavage rate k and the cleavage rate for substrate V: benzoyl-Ile-Glu-Gly-Arg-paranitroanilide k (I) / k (V), k (II) / k (V) , K (III) / k (V) and k (IV) / k (V) (these proportions are the same as the corresponding proportions measured for the substantially contaminating protease-free bovine coagulation factor Xa. (Or lower) can be assessed as being equal to or better than the substrate specificity of bovine blood coagulation factor Xa. Within the range characterized in this way, k (I) / k (V) is up to 0.06 and k (II) / k (V) is up to 0. It is more preferably 005. A serine protease type polypeptide as defined above will typically have a maximum molecular weight of 70,000 and a minimum of 15,000 molecular weight Mr. One such novel polypeptide according to the invention has the amino acid of SEQ ID NO: 2 and is an analog and / or homologue thereof. Another important embodiment of the polypeptide of the invention has an amino acid sequence that is a subsequence of SEQ ID NO: 2 or an analog and / or homologue of such subsequence. The term "similar to a polypeptide encoded by a DNA sequence" or "similar to a polypeptide having an amino acid sequence" means any polypeptide capable of acting as bovine coagulation factor Xa in the above test. .. Thus, polypeptides from different sources, such as different mammals or vertebrates, that differ to some extent in amino acid composition or post-translational modifications (eg, glycosylation or phosphorylation) as compared to the artificial serine proteases described in the Examples are also included. Thus, the term "analog" is used herein to refer to non-significant changes that alter the amino acid sequence, such as extra amino acids. An amino acid composition similar to the characteristic amino acid sequence of SEQ ID NO: 2 derived from the artificial serine protease described in Example 5, which yields an artificial serine protease analog by deletion, site-specific mutation, insertion or combination thereof of acid). Alternatively, it is used to indicate a protein or polypeptide having a sequence. Thus, within this specification and claims, analogs (of polypeptides) may be one or a number, provided that the enzymatic activity with the above specificity is retained, as can be evaluated from the above. It indicates a mutation in a polypeptide in which an amino acid has been deleted or exchanged and / or an amino acid has been introduced. In terms of homology, the polypeptide analogs according to the invention result in deletions and / or insertions of up to 50 amino acid residues, at least 60% at the polypeptide level compared to the sequence of the fragment of SEQ ID NO: 2. Will have sequence homology of identity. Such a polypeptide sequence or analog thereof having at least 60% homology to the polypeptide set forth in SEQ ID NO: 2 encoded by the DNA sequence of the present invention of SEQ ID NO: 1 or its analogs and / or homologues , An important embodiment of the present invention. The term "sequence homology" means identity in the sequence of either an amino acid in a segment of two or more amino acids in an amino acid sequence, or a nucleotide in a segment of two or more nucleotides of a nucleotide sequence. Thus, with respect to polypeptides, the term means homology between amino acids in question for which homology is established in the balance of amino acid identity and position of the polypeptide. Therefore, the term "homologous" is used herein to indicate the degree of identity between the amino acid sequence of a given polypeptide and the amino acid sequence set forth in SEQ ID NO: 2. The amino acid sequence compared to the amino acid sequence shown in SEQ ID NO: 2 is derived from or derived from a nucleotide sequence such as the DNA or RNA sequence obtained by hybrid formation as defined below. It can be obtained by a conventional amino acid sequencing method. Another embodiment has an amino acid sequence in which a string of 20 amino acids is at least 40% homologous to a thread of an amino acid of the same length selected from the amino acid sequence set forth in SEQ ID NO: 2. Regarding polypeptides. One serine protease polypeptide according to the invention has the amino acid sequence of SEQ ID NO: 2, residues 82-484, or is an analog 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 analog and / or homologue thereof. The many sequence modifications shown here are of particular interest: A sequence that cleaves SEQ ID NO: 38 or SEQ ID NO: 40-42 in place of residues 230-233 in SEQ ID NO: 2, preferably combined with the exchange of cysteine residue 245 for Gly, Ser or Arg in SEQ ID NO: 2. Insertion. Another interesting possibility is the insertion of SEQ ID NOs: 38, or SEQ ID NOs: 40-42, instead of residues 179-182 in SEQ ID NO: 2. Very generally, in any of the artificial serine proteases defined above, this by replacing the cleavage sequence corresponding to residues 230-233 in SEQ ID NO: 2 with one of the cleavage sequences defined above. A very useful cleavage enzyme used in the method of the invention will be produced. This cleaving enzyme can be selectively and very efficiently cleaved by an enzyme having specific enzymatic activity of bovine coagulation factor Xa, and thus by the artificial serine proteases described above (including the same molecule). This is because the first molecule that is cleaved and activated can cleave other molecules and thereby initiate a chain reaction, thus providing an artificial serine protease that is modified by such insertion of a specific cleaved sequence. Means that it is activated very efficiently. As mentioned above, the ability of artificial serine proteases to be produced by recombinant DNA technology is a very important feature, so another important embodiment of the invention is to encode the polypeptide as described above. With respect to nucleic acid fragments capable of encoding, particularly DNA fragments capable of encoding artificial serine protease polypeptides as described above. In one of those aspects, the invention relates to a nucleotide sequence that encodes the polypeptide of the invention as defined above. In particular, the invention has at least 60% homology to either the nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 1 or the DNA sequence set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2. With respect to their analogs encoding polypeptides that are at least 60% homologous to the amino acid sequences shown in. General Specifically, only the coding region is used when comparing nucleotide sequences to determine internal homology. The term "analog" for a DNA fragment of the invention refers to a nucleotide sequence that encodes a polypeptide that is identical or substantially identical to the polypeptide encoded by the DNA fragment of the invention. It is known that the same amino acid is encoded by various codons and the use of the codon relates specifically to the preference of the organism in question to express the nucleotide sequence. Thus, one or more nucleotides or codons in the DNA segment of the invention may be exchanged for another that produces a polypeptide that is identical or substantially identical to the polypeptide encoded by the DNA fragment in question being expressed. it can. In addition, the term "analog" does not practically affect sequence mutations (polypeptides encoded by DNA fragments) such as substitutions, insertions (including introns), additions and rearrangements of one or more nucleotides. ) Is intended to be given. Thus, modified nucleotide sequences that differ from the DNA sequence shown in SEQ ID NO: 1 in which at least one nucleotide has been substituted, added, inserted, deleted and / or rearranged are included within the scope of the invention. The term "substitution" means replacing one or more nucleotides in the complete nucleotide sequence with one or more different nucleotides, and "addition" means one or more nucleotides at any end of the complete nucleotide sequence. "Insert" means to introduce one or more nucleotides into the complete nucleotide sequence, and "deletion" means that one or more nucleotides are in any of the sequences. "Relocation" means that two or more nucleotide residues have been deleted from the complete nucleotide sequence, either at the ends or at appropriate locations within it, within the DNA or polypeptide sequence. It means that it has been exchanged. But the DNA fragment inserts it into the organism It may be modified by mutagenesis either before or after. The DNA or protein sequence of the present invention is either a biophysical, biochemical or biological property, or a portion or part of such property (one and / or all) or such property (one and one and). It may be modified in such a way that it does not lose all / or all). Examples of specific analogs of the DNA sequences of the present invention include DNA sequences consisting of DNA sequences shown in SEQ ID NO: 1 and specifically adapted for expression in E. coli. This DNA sequence, when inserted into E. coli with the appropriate regulatory sequence, expresses a polypeptide having substantially the amino acid sequence set forth in SEQ ID NO: 2. Therefore, this DNA sequence consists of specific codons recognized by E. coli. The terms "fragment", "sequence", "homlogue" and "analog" as used herein and in the claims relating to fragments, sequences, homologues and analogs according to the invention are these in their natural environment. It should of course be understood as consisting of these phenomena in, for example, in vitro isolated, purified, or recombinant forms, rather than those of the above phenomena. One embodiment of the nucleic acid fragment according to the invention is that at least 60% of the decoding triplets are identical to the nucleic acid fragment of the nucleic acid encoding bovine coagulation factor X and encode the amino acid, with up to 150 nucleotide insertions and /. Alternatively, it is a nucleic acid fragment of the above definition that gives a deletion. Examples of such nucleic acid fragments include SEQ ID NO: 1, nucleotides 76-1527, and their analogs and / or homologues. Another example is SEQ ID NO: 1, nucleotides 319-1527, and their analogs and / or homologues. Yet another example is SEQ ID NO: 1, nucleotides 571-1527, and their analogs and / or homologues. The DNA fragments described above and which constitute an important aspect of the invention can be obtained directly from genomic DNA or It can be obtained by isolating mRNA and converting it to the corresponding DNA sequence using reverse transcriptase, thereby producing cDNA. When a DNA fragment is obtained from genomic DNA, the DNA fragment is directly derived by screening for genomic sequences known to those skilled in the art. It can be achieved by knowledge of the sequences of the present invention, or by hybrid formation to DNA probes designed based on sequence information obtained by the amino acid sequencing method of purified serine proteases. If the DNA is of complementary DNA (cDNA) origin, it is a cDNA library (cDNA) that uses mRNA from cells containing artificial serine proteases. It can be obtained by creating a library). Hybridization can be achieved with DNA probes designed based on knowledge of cDNA sequences or sequence information obtained from the amino acid sequences of purified artificial serine proteases. The DNA fragment of the invention or its analogs and / or homologues of the invention can be replicated by fusing it with a vector and inserting the complex into a suitable microbial or mammalian cell line. Alternatively, the DNA fragment can be produced using chemical synthesis. In addition, the polymerase chain reaction (PCR) primer can be synthesized based on the nucleotide sequence shown in SEQ ID NO: 1. These primers can then be used to amplify all or part of the sequence encoding the artificial serine protease polypeptide. Suitable polypeptides of the invention can be produced using recombinant DNA technology. More specifically, the polypeptide breeds an organism carrying the DNA sequence or analogs and / or homologues set forth in SEQ ID NO: 1 of the invention under conditions leading to the expression of the DNA fragment. It can then be produced by a method consisting of recovering the expressed polypeptide from the organism. The organism used to produce the polypeptide may be a higher organism such as an animal or a lower organism such as a microorganism. Regardless of the type of organism used, the DNA fragment of the invention (above) should be introduced into an organism either directly or with the help of a suitable vector. In addition, polypeptides can be introduced into mammalian cell lines either directly or with the help of expression vectors by introducing the DNA fragments of the invention, or analogs and / or homologues thereof. Can be produced. The DNA fragments of the invention can also be cloned into a suitable stable expression vector and then placed in a suitable cell line. You can also do it. Cells expressing the desired polypeptide are then selected using the appropriate conditions for the vector and cell line used. The selected cells are then further proliferated to form a very important and continuous source of the desired polypeptide. Thus, another aspect of the invention relates to an expression system consisting of the nucleic acid fragment of the above definition and encoding the artificial serine protease polypeptide of the above definition, wherein the system mediates the expression of the nucleic acid fragment. It consists of a 5'-flanking sequence that can be made. The expression system can be a replicable expression vector carrying nucleic acid flanking (which can replicate in a host organism or cell line). The vector may be, for example, a plasmid, phage, cosmid, minichromosome or virus, and the vector may be one that integrates into the host cell genome when introduced into the host cell. Another aspect of the invention relates to an organism capable of carrying and replicating a nucleic acid fragment as defined above. The organism can be a microorganism (eg, bacterium, yeast, protozoan), or a cell from a multicellular organism (eg, mold, insect cell, plant cell, mammalian cell) or cell lineage. Particularly interesting host organisms are microorganisms such as bacteria of the Escherichia, Bachillus or Salmonella species. Yet another aspect of the invention is that step 1 the nucleic acid fragment as defined above is inserted into an expression vector, 2 the host organism is transformed with the vector produced in step a, and 3 step b. The host organism produced is cultured to express the polypeptide, 4 the polypeptide is collected, 5 optionally the polypeptide is post-translationally modified, and 6 if necessary, the polypeptide is transformed / restored in the invention. Attached to the method, 7 Optionally, the present invention relates to a method for producing the above-mentioned serine protease polypeptide, which comprises subjecting the polypeptide to further modifications to obtain the above-defined authentic polypeptide. Further modification of the polypeptide can be achieved, for example, by subjecting the polypeptide to carboxypeptidase A or B, whereby the selected amino acid residue can be removed from the C-terminus of the polypeptide molecule. .. This is only achieved if optimal folding of the authentic polypeptide molecule is placed at the C-terminus of the authentic polypeptide (eg, SEQ ID NO: 37) where the N-terminus is free and therefore cleaved. It is desirable in such a situation. As is known, carboxypeptidase B cleaves continuously from the C-terminus and only cleaves basic amino acids, while carboxypeptidase A cleaves non-basic amino acids. By carefully designing which residues are adjacent to the C-terminus of the authentic amino acid, it is possible to ensure that all but the authentic polypeptide is cleaved by carboxypeptidase. If the C-terminus of the authentic polypeptide is a basic amino acid residue, it should be ensured that the C-terminus binding residue to be removed is non-salted, and vice versa. If the sequence of amino acid residues from the C-terminus to the C-terminus of the authentic polypeptide is known, it alternates between treatments with two carboxypeptidases until only the bare authentic polypeptide remains. be able to. In a practical embodiment, an immobilized carboxypeptidase is used. The polypeptide produced comprises one or more steps such as affinity chromatography and / or other chromatographic means and electrical perturbation means using an immobilized polypeptide or an antibody that reacts with the polypeptide. It can be isolated by the method. The polypeptide of the present invention can also be prepared by a known liquid phase or solid phase peptide synthesis method utilizing continuous coupling of individual amino acids of the polypeptide sequence. Will be understood. The polypeptide can also be synthesized by coupling individual amino acids that form fragments of the polypeptide sequence that will be coupled later so that the desired polypeptide is obtained. Thus, these methods constitute another interesting aspect of the invention. The present invention has an 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, the artificiality of the above definition for cleaving a polypeptide at the cleavage site of bovine coagulation factor Xa. Uses of serine protease polypeptides and modified amino acid sequences selected from the group SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 (cleaveable as further described above). It relates to the use of the artificial serine protease polypeptide as defined above for cleaving the polypeptide at the cleavage site of bovine coagulation factor Xa (converted to morphology). Explanatory drawing Figure 1: Schematic representation of a periodic degeneration / restoration time plan. The composition of the solvent is expressed by the relative ratio of the buffer solution B to the mixed solution of the two components of the non-denatured "buffer solution A" and the modified "buffer solution B". Three consecutive cycles are represented, each cycle consisting of a convalescent phase "F" and a denatured phase "D". The change in the level of denaturing power of the solvent mixture in the denaturing phase of continuous cycles is represented by "K". Figure 2: Expression plasmid pT Schematic representation of a periodic degeneration / restoration time plan. The composition of the solvent is expressed by the relative ratio of the buffer solution B to the mixed solution of the two components of the non-denatured "buffer solution A" and the modified "buffer solution B". Three consecutive cycles are represented, each cycle consisting of a convalescent phase "F" and a denatured phase "D". The change in the level of denaturing power of the solvent mixture in the denaturing phase of continuous cycles is represented by "K". Figure 2: Expression plasmid pT Schematic representation of a periodic degeneration / restoration time plan. The composition of the solvent is expressed by the relative ratio of the buffer solution B to the mixed solution of the two components of the non-denatured "buffer solution A" and the modified "buffer solution B". Three consecutive cycles are represented, each cycle consisting of a convalescent phase "F" and a denatured phase "D". The change in the level of denaturing power of the solvent mixture in the denaturing phase with continuous cycles is represented by "K". Figure 2: Expression plasmid pT₇H₆FX-hβ2m and pT₇H₆FX-mβ2m configuration. Amino acid residue Ile fused at the 5'end to the nucleotide sequence encoding the FXa cleavage site (SEQ ID NO: 37)₁From Met₉₉Amplified DNA fragments containing human and mouse β2-microglobulin reading frames up to are cleaved with restriction endophen nucleases Bam HI and Hind III (purchased from Boehringer, Germany) and T using standard procedures.₄Bam HI and Hind III cleavage pT by DNA ligase (purchased from Boehringer, Germany)₇H₆Combined with. Figure 3: Human and mouse β2-microglobulin amino acid sequences. A: Predicted amino acid sequence with a full-length reading frame that encodes human β2-microglobulin (SEQ ID NO: 49). Amino acid residues (Ile) in the treated mature protein are shown. B: Predicted amino acid sequence of full-length reading frame encoding mouse β2-microglobulin (SEQ ID NO: 50). Amino acid residues (Ile) in the treated mature protein are shown. Figure 4: Expression plasmid pT₇H₆Construction of FX-hGH. Amino acid residue Phe fused at the 5'end to the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38)₁From Phe₁₉₁Amplified DNA fragments containing a reading frame for human growth hormone up to are cleaved with restriction endophen nucleases Bam HI and Hind III (purchased from Boehringer, Germany) and T using standard procedures.₂Bam HI and Hind III cleavage pT by DNA ligase (purchased from Boehringer, Germany)₇H₆Combined with. Figure 5: Amino acid sequence of human growth hormone (somatotropin). Predicted amino acid sequence in full-length reading frame encoding human growth hormone (SEQ ID NO: 51). First amino acid residue of processed mature protein (Phe)₁)It is shown. Figure 6: Human α₂-Macroglobulin receptor protein (α)₂MR) (SEQ ID NO: 52) amino acid residue numbers 20 (Ala) to 109 (Arg), amino acid residue numbers 20 (Ala) to 190 (Ala), and amino acid residue numbers 20 (Ala) to 521 ( Plasmid pT expressing up to Lys)₇H₆FX- # 1, # 2 and # 3 configurations. # 1: Amino acid residue numbers 20 (Ala) to 109 (Arg), # 2: Amino acid residue numbers 20 (Ala) to 190 (Ala), and # 3: Amino acid residue numbers 20 (Ala) to 520. Α up to (Lys)₂Amplified DNA fragments derived from the MR reading frame and fused at the 5'end with the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38) were purchased from restricted nucleovas Bam HI and Hind III (Boehringer, Germany). ) And T using standard procedures₄Bam HI and Hind III cleavage pT by DNA ligase (purchased from Boehringer, Germany)₇H₆Combined with. Figure 7: Human α₂-Macroglobulin receptor protein (α)₂MR) (SEQ ID NO: 52) amino acid residue numbers 803 (Gly) to 1265 (Asp), amino acid residue numbers 849 (Val) to 1184 (Gln), and amino acid residue numbers 1184 (Gln) to 1582 ( The plasmid pLcIIMLCH that expresses up to Lys)₆FX- # 4, # 5 and # 6 configurations. # 4: Amino acid residue numbers 803 (Gly) to 1265 (Asp), # 5: Amino acid residue numbers 849 (Val) to 1184 (Gln), and # 6: Amino acid residue numbers 1184 (Gln) to 1582. Α up to (Lys)₂Amplified DNA fragments derived from the MR reading frame and fused at the 5'end with the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38) were purchased from restricted nucleovas Bam HI and Hind III (Boehringer, Germany). ) And T using standard procedures₄Bam HI and Hind III cleavage by DNA ligase (purchased from Boehringer, Germany) pLcIIMLCH₆Combined with FX. Figure 8: Human α₂-Macroglobulin receptor protein (α)₂MR) (SEQ ID NO: 52) amino acid residue numbers 803 (Gly) to 1582 (Lys), amino acid residue numbers 2519 (Ala) to 2941 (Ile), and amino acid residue numbers 3331 (Val) to 3778 ( Plasmid pLcIIMLCH expressing up to Ile)₆FX- # 7, # 8 and # 9 configurations. # 7: Amino acid residue numbers 803 (Gly) to 1582 (Lys), # 8: Amino acid residue numbers 2519 (Ala) to 2941 (Ile), and # 9: Amino acid residue numbers 3331 (Val) to 3778. Α up to (Ile)₂Amplified DNA fragments derived from the MR reading frame and fused at the 5'end with the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38) were purchased from restricted nucleovas Bam HI and Hind III (Boehringer, Germany). ) And T using standard procedures₄Bam HI and Hind III cleavage by DNA ligase (purchased from Boehringer, Germany) pLcIIMLCH₆Combined with FX. Figures 9a and 9b: Human α₂-Macroglobulin receptor protein (α)₂Amino acid sequence of MR) (SEQ ID NO: 52). α₂Predicted amino acid sequence of full-length reading frame that encodes MR. Amino acid residues present as N- or C-terminus in recombinant protein are α₂It is identified by the number on the MR sequence. Figure 10: Expression plasmid pLcIIMLCH₆Composition of FX-FXΔγ. Amino acid residue Ser fused at the 5'end to the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38)₈₂From Trp₄₈₄Amplified DNA fragments containing a reading frame for bovine blood coagulation factor X up to are cleaved with restricted nucleovas Bam HI and Hind III (purchased from Boehringer, Germany) and T using standard procedures.₄Bam HI and Hind III cleavage by DNA ligase (purchased from Boehringer, Germany) pLcIIMLCH₆Combined with FX. Figure 11: Amino acid sequence of bovine blood coagulation factor X (FX). Predicted amino acid sequence of full-length reading frame encoding bovine FX (SEQ ID NO: 53). N-terminal amino acid residue Ser of FXΔγ construct₈₂And C-terminal residue Trp₄₈₄Has been identified. Figure 12: Expression plasmid pLcIIMLCH₆FX-K1 configuration. Amino acid residue Ser fused at the 5'end to the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38)₈₂From Glu₁₆₂Amplified DNA fragments containing a reading frame for human plasminogen Kringle 1 (K1) up to ("Glu"-numbered as in plasminogen) were purchased from restricted endophen nucleases Bam HI and Hind III (Boehringer, Germany). Cut using T₄Bam HI and Hind III cleavage by DNA ligase (purchased from Boehringer, Germany) pLcIIMLCH₆Combined with FX. Figure 13: Expression plasmid pLcIIH₆FX-K4 configuration. Amino acid residue Val fused at the 5'end to the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38)₃₅₄From Ala₄₃₉Amplified DNA fragments containing a reading frame for human plasminogen Kringle 4 (K4) up to ("Glu"-numbered as in plasminogen) were purchased from restricted endophen nucleases Bam HI and Hind III (Boehringer, Germany). Cut using T₄Bam HI and Hind III cleavage by DNA ligase (purchased from Boehringer, Germany) pLcIIH₆Combined with FX. Figure 14: Amino acid sequence of human "Glu"-plasminogen (SEQ ID NO: 54). The N-terminal and C-terminal amino acid residues in the K1 and K2 constructs have been identified by their numbers in the sequence. Figure 15: Recombinant human β₂-SDS-PAGE analysis of microglobulin production and in vitro folding. Lane 1: Ni²⁺Crude protein extract (reduced sample) before application to NTA-agarose column. Lane 2: Crude protein extract Ni²⁺NTA-flow-through (reduced sample) of the column while applying to the agarose column. Lane 3: Ni with non-denaturing elution buffer after periodic folding steps²⁺Human β eluted from NTA-Agarose column₂-Microglobulin (reduced sample). Lane 4: Protein markers (Pharmacia, Sweden): From the top of the gel; 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa and 14.4 kDa (reduced samples). Lane 5: Same as Lane 3 (non-reduced sample). Lane 6: Recombinant human β after FXa cleavage and final purification₂-Microglobulin (non-reducing sample). Figure 16: Recombinant human growth hormone; SDS-PAGE analysis of hGH (somatotropin) in vitro folding. Lane 1: Protein markers (Pharmacia, Sweden): From the top of the gel; 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa and 14.4 kDa (reduced samples). Lane 2: Ni by non-denaturing elution buffer after periodic folding process²⁺NTA-Human hGH (non-reducing sample) eluted from an agarose column. Lane 3: Ni by denatured elution buffer B from the folding process after the periodic folding process²⁺NTA-Human hGH (non-reducing sample) eluted from an agarose column. Lanes 4-18: Collected during separation of monomeric hGH fusion proteins from dimeric and multimeric fusion proteins by ion exchange chromatography with Q-Sepharose (Pharamacia, Sweden) after a periodic folding step. Fraction that was done. The monomeric protein was eluted as a peak completely separated from the peak containing dimeric and multimeric proteins (non-reducing sample). Figure 17: Recombinant Kringle 1 and 4 from human plasminogen, and human α₂-Macroglobulin receptor protein (α)₂SDS-PAGE analysis of in vitro folding of recombinant fusion protein # 4 from MR). Lane 1: Protein markers (Pharmacia, Sweden): From the top of the gel; 94 kDa, 67 kDa, 43 kDa, 30 kDa, 20.1 kDa and 14.4 kDa (reduced samples). Lane 2: Ni²⁺Crude K1-fusion protein extract (reduced sample) before application to NTA-agarose column. Lane 3: Ni by non-denaturing elution buffer after periodic folding process²⁺K1-fusion protein eluted from NTA-agarose column (reduced sample). Lane 4: Same as Lane 3: (non-reduced sample). Lane 5: Flow-through (non-reducing sample) from a lysine-agarose column while applying the K1-fusion protein. Lane 6: K1-fusion protein eluted from a lysine agarose column (non-reducing sample). Lane 7: Ni by non-denaturing elution buffer after periodic folding process²⁺K4-fusion protein (reduced sample) eluted from the NTA-agarose column. Lane 8: Same as Lane 7 (non-reduced sample). Lane 9: Ni by non-denaturing elution buffer after periodic folding process²⁺NTA-α eluted from the agarose column₂MR # 4 fusion protein (reduced sample). Lane 10: Same as Lane 9 (non-reducing sample). Figure 18: Expression plasmid pT₇H₆FX-α₂MRBDv configuration. Amino acid residue Val fused at the 5'end to the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38)₁₂₉₉From Ala₁₄₅₁Up to human α₂-Amplified DNA fragments containing macroglobulin reading frames are cleaved with restricted endon nucleases Bam HI and Hind III (purchased from Boehringer, Germany) and T using standard procedures.₄It bound to Bam HI and HindIII cleavage pT7H6 by DNA ligase (purchased from Boehringer, Germany). Figure 19: Human α₂-Amino acid sequence of the receptor binding domain of macroglobulin (residue Val)₁₂₉₉From Ala₁₄₅₁Up to) (SEQ ID NO: 55). Figure 20: Expression plasmid pT₇H₆FX-TETN configuration. Amino acid residue Glu fused at the 5'end to the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38)₁From Val₁₈₁Amplified DNA fragments containing a reading frame of mature monomeric human Tetranectin up to (purchased from Boehringer, Germany) were cleaved with restricted endon nucleases Bam HI and Hind III (purchased from Boehringer, Germany) and T using standard procedures.₄Bam HI and Hind III cleavage pT by DNA ligase (purchased from Boehringer, Germany)₇H₆Combined with. Figure 21: Amino acid sequence of human monomeric tetranectin. Predicted amino acid sequence of full-length reading frame encoding human tetranectin (SEQ ID NO: 56). First amino acid residue of processed mature protein (Glu)₁)It is shown. Figure 22: Expression plasmid pT₇H₆FX-DB32 configuration. Amino acid residue Gln fused at the 5'end to the nucleotide sequence encoding the FXa cleavage site IEGR (SEQ ID NO: 38)₁From Asn₂₄₆Amplified DNA fragments containing the reading frame of the artificial diabodies DB32 up to were cleaved with restricted endon nucleases Bam HI and Hind III (purchased from Boehringer, Germany) and T using standard procedures.₄Bam HI and Hind III cleavage pT by DNA ligase (purchased from Boehringer, Germany)₇H₆Combined with. Figure 23: Amino acid sequence of artificial diabodies DB32 (SEQ ID NO: 57). Figure 24: Expression plasmid pT₇H₆FX-PS.4. Amino acid residue Ser₂From Gln₁₀₁PT expressing up to human saliasin (psoriasin)₇H₆The configuration of FX-PS.4 has already been described (Hoffmann, 1994). Figure 25: Amino acid sequence of human saliacin. Predicted amino acid sequence of full-length reading frame encoding human saliacin (SEQ ID NO: 58). Figure 26: Purification of recombinant Mab32 diabodies and SDS-PAGE analysis of FXa cleavage. A: Different stages of purification. Lane 1 and Lane 2: Crude protein from folding. Lane 3: Final Purified Mab32 Diabody Fusion Protein Product. Lane 4: Supernatant of coarsely folded product after 50-fold concentration and centrifugation. Lane 5: Pellets from coarsely folded products after 50x concentration and centrifugation. b: FXa cleavage of Mab32 diaboly fusion protein. Lanes 1 and 5: Final Purified Mab32 Diabody Fusion Protein. Lane 2: 37 ° C 20 hours Mol rate 1: 5 FXa: Mab32 Diabody fusion protein. Lane 3: 37 ° C 20 hours Mol rate 1: 2 FXa: Mab32 Diabody fusion protein. Lane 4: 37 ° C 20 hours Mol rate 1: 1 FXa: Mab32 Diabody fusion protein. Figure 27: Human β₂-Appropriateness of glutathione as a reducing agent in the periodic regeneration of microglobulin fusion proteins. Lane 1: Reduction sample of test number 1. Lane 2: Non-reduced sample with test number 1. Lane 3: Non-reduced sample with test number 2. Lane 4: Non-reduced sample with test number 3. Lane 5: Non-reduced sample with test number 4. Lane 6: Non-reduced sample with test number 5. Lane 7: Non-reduced sample with test number 6. Lane 8: Non-reduced sample with test number 7. Lane 9: Non-reduced sample with test number 8. Lane 10: Non-reduced sample with test number 9. Lane 11: Non-reduced sample with test number 10. Lane 12: Non-reduced sample with test number 11. Figure 28: Human β₂-Appropriateness of L-Cysteine Ethyl Ester as a Reducing Agent in the Periodic Regeneration of Microglobulin Fusion Proteins. Lane 1: Reduction sample of test number 1. Lane 2: Non-reduced sample with test number 1. Lane 3: Non-reduced sample with test number 2. Lane 4: Non-reduced sample with test number 3. Lane 5: Non-reduced sample with test number 4. Lane 6: Non-reduced sample with test number 5. Lane 7: Non-reduced sample with test number 6. Lane 8: Non-reduced sample with test number 7. Lane 9: Non-reduced sample with test number 8. Lane 10: Non-reduced sample with test number 9. Figure 29: Human β₂-Appropriateness of 2-mercaptoethanol as a reducing agent in the periodic regeneration of microglobulin fusion proteins. Lane 1: Reduction sample of test number 1. Lane 2: Non-reduced sample with test number 1. Lane 3: Non-reduced sample with test number 2. Lane 4: Non-reduced sample with test number 3. Lane 5: Non-reduced sample with test number 4. Lane 6: Non-reduced sample with test number 5. Lane 7: Non-reduced sample with test number 6. Lane 8: Non-reduced sample with test number 7. Lane 9: Non-reduced sample with test number 8. Lane 10: Non-reduced sample with test number 9. Figure 30: Human β₂-Appropriateness of mercaptosuccinic acid as a reducing agent in the periodic regeneration of microglobulin fusion proteins. Lane 1: Non-reduced sample with test number 1. Lane 2: Non-reduced sample with test number 2. Lane 3: Non-reduced sample with test number 3. Lane 4: Non-reduced sample with test number 4. Lane 5: Non-reduced sample with test number 5. Lane 6: Non-reduced sample with test number 6. Lane 7: Non-reduced sample with test number 7. Lane 8: Non-reduced sample with test number 8. Lane 9: Non-reduced sample with test number 9. Figure 31: Human β₂-Appropriateness of N-Acetyl-L-Cysteine as a reducing agent in the periodic regeneration of microglobulin fusion proteins. Lane 1: Reduction sample of test number 1. Lane 2: Non-reduced sample with test number 1. Lane 3: Non-reduced sample with test number 2. Lane 4: Non-reduced sample with test number 3. Lane 5: Non-reduced sample with test number 4. Lane 6: Non-reduced sample with test number 5. Lane 7: Non-reduced sample with test number 6. Lane 8: Non-reduced sample with test number 7. Lane 9: Non-reduced sample with test number 8. Lane 10: Non-reduced sample with test number 9. Figure 32: Human β₂-SDS-PAGE analysis of periodic regeneration of microglobulin fusion proteins. Lane 1: Ni²⁺Crude protein extract (reduced sample) before application to NTA-agarose column. Lane 2: 8 μl sample of the soluble fraction of regenerated hβ2 m described in Example 1. Lane 3: 4 μl sample of the soluble fraction of regenerated hβ2 m described in Example 1. Lane 4: 2 μl sample of the soluble fraction of regenerated hβ2 m described in Example 1. Lane 5: 8 μl sample of the insoluble fraction of regenerated hβ2 m described in Example 1. Lanes 6 and 7: hβ2m end product after purification by ion exchange chromatography. Lanes 8 and 9: Regeneration hβ2m after the optimal regeneration protocol described in Example 13. Figure 33: Human β with buffer step and linear gradient₂-SDS-PAGE analysis of microglobulin fusion protein regeneration. Lane 1: Samples from the soluble fraction of regenerated hβ2m folded by the buffer step protocol described in Example 13. Lane 2: Samples from the insoluble fraction of regenerated hβ2m folded by the buffer step protocol described in Example 13. Lane 4: Protein Molecular Weight Marker (Pharmacia, Sweden): From the top of the gel; 94kDa, 67kkDa, 43kDa, 30kDa, 20.1kDa and 14.4kDa (reduced sample). Lane 5: Samples from the soluble fraction of regenerated hβ2m folded by the linear gradient protocol described in Example 13. Lanes 6 and 7: Samples from the insoluble fraction of regenerated hβ2m folded by the linear gradient protocol described in Example 13. Figure 34: A general schematic of the design of the fusion proteins described in the Examples. At the N-terminus of the fusion protein, a "booster segment" that enhances the expression level of the fusion protein in cells expressing the DNA encoding the fusion protein is optionally inserted. At the C-terminus of this, the "6H" is the "affinity" during purification and regeneration of the fusion protein. The six histidinel residues that make up the ion chelate site used as "handle)" are shown. The C-terminal "FX" of the 6 histidinel sites is the FXa cleavage site. Finally, the portion of the fusion protein labeled "protein" represents the protein that will be regenerated according to the method of the invention. Examples All of the examples 1 to 11 in this section, used to illustrate the "periodic folding step", are produced in E. coli, purified from crude protein extracts, and general without further purification. The folding process of a cleavable recombinant hybrid protein (fusion protein) that can be folded by a conventional procedure is described. The nucleotide sequence that encodes the recombinant protein to be produced is fused at the 5'-terminus to the nucleotide sequence that encodes the amino acid sequence that identifies the FXa cleavage site (FX), followed by a segment containing 6 histidinyl residues ( It is attached to the N-terminus of SEQ ID NO: 47). Binding of the FXa cleavage site is usually achieved during a polymerase chain reaction in which the 5'-terminal primer consists of nucleotides encoding this sequence. Binding of 6 histidine residues is usually obtained by using a vector consisting of a nucleotide fragment encoding SEQ ID NO: 47. The six histidine residues constitute a metal ion chelate site that is used as an affinity handle during the purification of the fusion protein and subsequently as a point of contact with the solid matrix during the periodic folding process. Occasionally, a "booster segment" (eg, a segment from the N-terminus of the λcII protein, sometimes followed by a segment from the myosin L chain) is an affinity handle to improve the expression level of the fusion protein in E. coli. Is inserted at the N-terminus of. All fusion proteins are designed according to the same general schematic (see Figure 34). The presence of booster segments, affinity handles and FXa cleavage sites may complicate the regeneration of the recombinant protein of interest. In addition, the cycle The folding process is initiated immediately after purification by the affinity of the fusion protein. This means that the fusion protein material partially degraded by the host E. coli remains on the affinity matrix in addition to the full length fusion protein column. This degraded fusion protein will significantly interfere with the regeneration of the full-length fusion protein, thereby reducing the apparent efficiency of the process. Therefore, the folding efficiencies reported in Examples 1 to 11 cannot be directly compared to the efficiencies of the purified fusion protein regeneration step. Examples 1 to 11 range in size from 82 amino acids (K1, Example 6) to 780 amino acids (α).₂The regeneration steps of 21 different proteins, protein domains or domain clusters up to MR # 7, Example 4) are described, and the range of the number of disulfide bridges in the protein is zero (α).₂MRAP, Examples 3) to 33 (α)₂MR # 4, Example 4) and 36 (α)₂MR # 7, Example 4). The efficiency of protein regeneration ranges from 15% to 95%, and the yield of active protein is 40 ml Ni.⁺Regeneration with an NTA-agarose column (NTA stands for substituted nitrilotriacetic acid) is on the order of 10-100 mg. Table from the next 1 to 5, graph used in Example illustrates the di-entry profile. "Hour" is in minutes and "flow" is in ml / min.<img file="JP3695467B2_D0003.tif" /><img file="JP3695467B2_D0004.tif" /><img file="JP3695467B2_D0005.tif" /><img file="JP3695467B2_D0006.tif" /><img file="JP3695467B2_D0007.tif" />Example 1 Human and mouse β₂-Microglobulin production and folding This example shows human β as an FXa cleavable fusion protein.₂-Microglobulin and mouse β₂-Production of both microglobulins in E. coli, and recombinant β in humans and mice after FXa cleavage₂-Describes purification of microglobulin. Human and mouse β₂-Plasmid clones containing full-length cDNA encoding microglobulin proteins (provided to Dr. Sφren Buus in favor of Dr. David N. Garboczi), SEQ ID NO: 3 and SEQ ID NO: 4 (human β).₂-For microglobulins) and SEQ ID NO: 5 and SEQ ID NO: 6 (mouse β)₂-Mature human β with primers (for microglobulin)₂-Microglobulin protein (amino acid residue Ile₁From Met₉₉Equivalent to) and mature mouse β₂-Microglobulin protein (amino acid residue Ile₁From Met₉₉Used as a template in the polymerase chain reaction (PCR) designed to produce the cDNA fragment corresponding to (Saiki et al., 1988). The amplified code reading frame was subjected to PCR reaction at the 5'end to obtain the amino acid sequence of SEQ ID NO: 37 (Nagai and Thφgersen, 1987) contained in SEQ ID NOs: 3 and 5 and constituting the cleavage site of bovine limiting protease FXa. Concatenated to the nucleotide sequence to encode. The amplified DNA fragment is expressed in E. coli expression vector pT.₇H₆Subcloned into (Christensen et al., 1991). Obtained plasmid pT₇H₆FX-hβ₂m (human β₂-Express microglobulin) and pT₇H₆FX-mβ₂m (mouse β₂The composition of -expressing microglobulin) is shown in Figure 2 and the amino acid sequence of the expressed protein is shown in Figure 3 (full length readings in SEQ ID NO: 49 (human) and SEQ ID NO: 50 (mouse)). The amino acid sequence encoded by the frame is shown.) Human and mouse β₂-Plasmid pT in E. coli BL21 cells on a medium scale (2 x 1 liter) as described in Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986.₇H₆FX-hβ₂m and pT₇H₆FX-mβ₂It was produced by growing and expressing m. Cultures that grow exponentially at 37 ° C, OD₆₀₀ At 0.8, the bacteriophage λCE6 was infected with a multiplicity of infection of about 5. After growing the culture at 37 ° C for an additional 3 hours, cells were collected by centrifugation. Cells were lysed by osmotic shock and sonication and all cell proteins were extracted into phenol (adjusted to pH 8 with trizuma base). The protein was precipitated from the phenol phase by the addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride and 50 mM tris hydrochloride pH 8 and 0.1 M dithioerythriol. Fusion protein after gel filtration into 8M urea, 1M sodium chloride, 50mM tris hydrochloride pH8, 10mM 2-mercaptoethanol and 3mM methionine using Sephadex G-25 (Sweden Pharmacia, LKB). That is, MGSHHHHHHGSIEGR-human and mouse β, respectively.₂-For purification of microglobulins (where MGSHHHHHHGSIEGR is SEQ ID NO: 48) (Hochuli et al., 1988), crude protein preparations, Ni²⁺The NTA-agarose column was applied to the activated NTA-agarose column, and the process was subsequently transferred to a periodic folding process. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Ni²⁺Activated by NTA-Agarose Matrix (Ni)²⁺NTA-agarose) is commercially available from Diagen GmbH of Germany. However, in the course of this study, it was found that this commercial product did not work as expected. According to our observation, commercially available Ni²⁺The NTA-agarose matrix was easily blocked when applied to the denatured and reduced total protein extract, the fusion protein capacity was lower than expected, and the matrix was successfully regenerated only a few times. .. Ni²⁺N- (5-amino-1-carboxypentyl) iminodiacetic acid metal ligand {synthetic route to a more rigid agarose matrix (ie, Sepharose CL-6B from Pharmacia, Sweden) to improve the performance of NTA-agarose. Decided to carbodiimide-couple} as described in Dobeli and Hochuli (EPO 0253 303). A solution containing 8 g of N- (5-amino-1-carboxypentyl) iminodiacetic acid obtained from the synthesis step in 50 ml was prepared to pH 10 by adding 29 g of sodium carbonate (10 hydrate) to 1 M. It was added to a stirred suspension containing activated Sepharose CL-6B in sodium carbonate. The reaction took place overnight. Sepharose CL-6B (initially a 100 ml suspension) was dehydrated and then activated by stirring with 7 g of acetone containing 1,1'-carbonyldiimidazole for 15 to 30 minutes. After activation, Sepharose CL-6B was washed with acetone and subsequently with water and 1 M sodium carbonate. The column is filled with this NTA-agarose matrix, and Ni is slowly passed through a 10% nickel sulfate solution for 5 column volumes.²⁺Was "loaded". Ni on the NTA-agarose matrix prepared in this step²⁺When the amount of was quantified, it was 14 μmol per 1 ml of matrix. This Ni²⁺The NTA-agarose matrix was packed in a standard class column for liquid chromatography (inner diameter 2.6 cm) in a volume of 40 ml. After loading, this Ni before applying the crude protein extract²⁺The NTA-agarose column was washed with 2 column volumes of water and 1 column volume of 1M Tris hydrochloride pH 8 and 2 column volumes of loading buffer. Ni²⁺After applying the crude protein extract to the NTA-agarose column, fusion proteins, ie MGSHHHHHHGSIEGR-hβ respectively₂m and MGSHHHHHHGSIEGR-mβ₂m (where MGSHHHHHHGSIEGR is SEQ ID NO: 48) is column-eluted with 1 column volume of loading buffer, followed by 6 M guanidinium chloride, 50 mM Tris hydrochloride, 10 mM 2-mercaptoethanol and 3 mM methionine. The solution was purified from most E. coli and λ phage proteins by washing until the solubility (OD) at 280 nm was stable. The fusion protein was prepared using 0.5 M sodium chloride and 50 mM Tris hydrochloride pH 8 and 1.2 mM / 0.4 mM reduced / oxidized glutathione as buffer A, using a gradient management profile as described in Table 1. Ni with 8M urea, 0.5M sodium chloride, 50 mM Tris hydrochloride pH 8 and 3 mM methionine and 6 mM reduced glutathione as buffer B.²⁺Regenerated on an NTA-agarose column. The reduced / oxidized glutathione solution was newly prepared as a 200-fold storage solution by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione prior to addition to buffer A. After the periodic folding process is completed, hβ₂m and mβ₂The m fusion protein was added to Ni using a buffer containing 0.5 M sodium chloride, 50 mM Tris hydrochloride and 20 mM EDTA pH 8.²⁺Eluted from NTA-agarose column. Ni²⁺The fusion protein that aggregated and precipitated on the NTA-agarose column was eluted in buffer B. Approximately 75% of the fusion protein material was eluted with a non-denaturing elution buffer (see lanes 2 and 3 in Figure 16). Judging from non-reducing SDS-PAGE analysis, about 70% soluble hβ₂m fusion protein material (40 mg hβ₂The m fusion protein (corresponding to the m fusion protein) appears to be monomeric (see lanes 5 and 3 in Figure 15), while 25% mβ.₂The m fusion protein appeared to be monomeric (20 mg mβ₂(equivalent to m fusion protein). Therefore, the overall efficiency of the folding process is hβ.₂About 50% for m fusion protein, mβ₂Less than 20% for m fusion proteins. Monomeric hβ₂m and mβ₂The m fusion protein was purified from dimers and higher multimers by ion exchange chromatography using S-Sepharose (Pharmacia, Sweden). The fusion protein (about 70% fusion protein material) eluted with a non-denatured elution buffer is gel filtered using Sephadex G-25 into a buffer containing 5 mM sodium chloride and 5 mM Tris hydrochloride pH 8. , Diluted 1: 1 with water before applying to the S-Sepharose ion exchange column. The fusion protein was eluted over 5 column volumes with a linear gradient from 2.5 mM sodium chloride and 2.5 mM tris hydrochloride pH 8 to 100 mM sodium chloride and 25 mM tris hydrochloride pH 8. Monomeric hβ₂m and mβ₂The m fusion protein was eluted at the very beginning of the gradient. On the other hand, dimers and higher multimers were eluted later. Fractions containing the monomeric fusion protein were diluted with water, reloaded onto an S-Sepharose column and eluted in one step with 1 M sodium chloride and 50 mM Tris hydrochloride pH 8. The monomeric fusion protein was cleaved with the limiting protease FXa at a weight ratio of about 200: 1 overnight at room temperature. After cleavage, recombinant hβ₂m and mβ₂The m protein was purified from the cleaved fusion protein and the N-terminal fusion tail free from FXa by ion exchange chromatography using a Q-Sepharose column (Pharmacia, Sweden). Recombinant hβ after gel filtration into 5 mM sodium chloride and 5 mM Tris hydrochloride pH 8 with Sephadex G-25 and 1: 1 dilution with water₂m and mβ₂m was eluted with a linear gradient (over 5 column volumes) from 2.5 mM sodium chloride and 2.5 mM Tris hydrochloride pH 8 to 100 mM sodium chloride and 25 mM Tris hydrochloride pH 8. Fractions containing the cleaved recombinant protein were diluted with water, reloaded onto a Q-Sepharose column and eluted in one step with 1 M sodium chloride and 50 mM Tris hydrochloride pH 8. Recombinant hβ₂m and mβ₂The m-protein was gel filtered into freshly prepared 20 mM ammonium bicarbonate and lyophilized twice. Recombinant human β₂-SDS-PAGE analysis of microglobulin production is shown in Figure 15. Fully treated recombinant human β produced by this step₂-The yield of microglobulin was 30 mg. Fully treated recombinant mouse β produced by this step₂-The yield of microglobulin was 10 mg. Recombinant human β₂-Microglobulin and purified natural human β₂-Comparison of microglobulins was kindly made by Dr. Sφren Buus by two different assays. 1. Recombinant human β₂-Microglobulin and natural human β₂-Microglobulins have been found to react with both monoclonal and monospecific antibodies with the same affinity. 2. Recombinant human β₂-Microglobulin and natural human β₂-Microglobulins are heavy chain class I K purified by natural affinity in binding inhibition experiments with radiolabeled ligands.^dIt was found that the molecules were bound with the same affinity. Recombinant mouse β₂-Microglobulin is a natural class I heavy chain molecule, human β₂-It was found to bind with a 5-fold lower affinity than microglobulin. This result is in good agreement with conventional results in the literature using natural materials. Example 2 Production and Folding of Human Growth Hormone (Somatotropin) This Example describes the production of human growth hormone (hGH) as an FXa cleavable fusion protein in E. coli and the purification of recombinant hGH after FXa cleavage. .. A plasmid clone {generally provided by Dr. Henrik Dalbφge (Dalbφge et al., 1987)} containing a cDNA that encodes hGH was used to mature hGH protein with primers of SEQ ID NO: 7 and SEQ ID NO: 8 (Dalbφge et al., 1987). Amino acid residue Phe₁From Phe₁₉₁Used as a template in the polymerase chain reaction (PCR) designed to produce the cDNA fragment corresponding to (Saiki et al., 1988). The amplified code reading frame encodes the amino acid sequence of SEQ ID NO: 37 (Nagai and Thφgersen, 1987) contained in SEQ ID NO: 7 and constituting the cleavage site of bovine limiting protease FXa by PCR reaction at the 5'end. It was linked to a nucleotide sequence. The amplified DNA fragment is expressed in E. coli expression vector pT.₇H₆Subcloned into (Christensen et al., 1991). Obtained plasmid pT₇H₆The composition of FX-hGH (expressing human growth hormone) is shown in Figure 4, and the amino acid sequence of the expressed protein is shown in Figure 5 (SEQ ID NO: 51 shows the amino acids encoded by the full-length reading frame. The sequence is shown.) Recombinant human growth hormone was added to Escherichia coli BL21 cells on a medium scale (2 x 1 liter) as described in Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986. In the plasmid pT₇H₆It was produced by growing and expressing FX-hGH. Cultures that grow exponentially at 37 ° C, OD₆₀₀ At 0.8, the bacteriophage λCE6 was infected with a multiplicity of infection of about 5. After growing the culture at 37 ° C for an additional 3 hours, cells were collected by centrifugation. Cells were lysed by osmotic shock and sonication and all cell proteins were extracted into phenol (adjusted to pH 8 with trizuma base). Protein was precipitated from the phenol phase by the 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 hydrochloride pH 8 and 50 mM dithioerythriol. Fusion protein after gel filtration into 8M urea, 1M sodium chloride, 50 mM Tris hydrochloride pH 8, 5 mM 2-mercaptoethanol and 1 mM methionine using Sephadex G-25 (Sweden Pharmacia, LKB). , That is, for purification of MGSHHHHHHGSIEGR-hGH (where MGSHHHHHGSIEGR is SEQ ID NO: 48) (Hochuli et al., 1988), a crude protein preparation, Ni.²⁺Activated by NTA-Agarose Column (Ni)²⁺It was applied to NTA-agarose) and subsequently moved to a periodic folding process. Ni²⁺The preparation and "loading" of the NTA-agarose column is described in Example 1. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Ni²⁺After applying the crude protein extract to the NTA-agarose column, the fusion protein, namely MGSHHHHHHGSIEGR-hGH (where MGSHHHHHHGSIEGR is SEQ ID NO: 48), was combined with a 1 column volume of loading buffer followed by 6M guanidinium chloride. Purified from most E. coli and λ phage proteins by washing the eluate with 50 mM Tris hydrochloride, 5 mM 2-mercaptoethanol and 1 mM methionine until the eluate was stable at 280 nm absorbance (OD). The fusion protein was prepared using 0.5 M sodium chloride and 50 mM Tris hydrochloride pH 8 and 1.0 mM / 0.1 mM reduced / oxidized glutathione as buffer A, using a gradient management profile as described in Table 2. Ni with 8 M urea, 0.5 M sodium chloride, 50 mM Tris hydrochloride pH 8 and 1 mM methionine and 5 mM reduced glutathione as buffer B.²⁺Regenerated on an NTA-agarose column. The reduced / oxidized glutathione solution was newly prepared as a 200-fold storage solution by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione prior to addition to buffer A. After the periodic folding process is complete, the hGH fusion protein is added to Ni with a buffer containing 0.5 M sodium chloride, 50 mM Tris hydrochloride and 20 mM EDTA pH 8.²⁺Eluted from NTA-agarose column. Ni²⁺The fusion protein that aggregated and precipitated on the NTA-agarose column was eluted in buffer B. Approximately 80% of the fusion protein material was eluted with a non-denaturing elution buffer (see lanes 2 and 3 in Figure 16). Judging from non-reducing SDS-PAGE analysis, about 90% of the soluble fusion protein material (corresponding to about 70 mg of fusion protein) appears to be monomeric (see lane 2 in Figure 16) and during the folding process. The overall efficiency was about 70%. The monomeric hGH fusion protein was purified from dimers and higher multimers by ion exchange chromatography using Q-Sepharose (Pharmacia, Sweden). After gel filtration with Sephadex G-25 into a buffer containing 25 mM sodium chloride and 25 mM Tris hydrochloride pH 8, the fusion protein material eluted with the non-denatured buffer is applied to the Q-Sepharose ion exchange column. did. The fusion protein was eluted over 5 column volumes with a linear gradient from 25 mM sodium chloride and 25 mM Tris hydrochloride pH 8 to 200 mM sodium chloride and 50 mM Tris hydrochloride pH 8. The monomeric hGH fusion protein was eluted at the very beginning of the gradient. On the other hand, the dimer and higher multimers were eluted later. Nickel sulfate and iminodiacetic acid (IDA, adjusted to pH 8 with sodium hydroxide) to a fraction containing a pure monomeric fusion protein up to 1 mM and using the limiting protease FXa at a weight ratio of approximately 100: 1 5 It was cut at 37 ° C for an hour. FXa is inhibited by the addition of benzamidine hydrochloride up to 1 mM after cleavage. After cutting, Ni²⁺Gel filtration into 8M urea and 50 mM Tris hydrochloride pH 8 with Sephadex G-25 to remove IDA and benzamidine, then completely FX respectively_aAnd Ni²⁺And Nd³⁺To remove the small Ni²⁺NTA-Agarose column followed by a small Nd in a row³⁺NTA-Agarose column and subsequently Ni²⁺Recombinant hGH protein was isolated from the uncleaved fusion protein and the free fusion tail by passing through an NTA agarose column that was not activated by. Recombinant hGH was purified from trace fractions of recombinant degradation products by ion exchange chromatography using Q-Sepharose. hGH was eluted with a linear gradient (over 5 column volumes) from 8M urea and 50 mM Tris hydrochloride pH 8 to 8 M urea and 250 mM sodium chloride and 25 mM Tris hydrochloride pH 8. Fractions containing the cleaved purified recombinant protein were gel filtered into freshly prepared 20 mM ammonium bicarbonate and lyophilized twice. SDS-PAGE analysis of recombinant human growth hormone production and folding is shown in Figure 16. The yield of fully treated recombinant human growth hormone produced by this step was 10 mg. Recombinant human growth hormone produced by this step is Novo-Nordisk in both reducing and non-reducing SDS-PAGE and non-denaturing PAGE analysis. It showed the same electrophoresis as the biologically active recombinant human growth hormone provided generously by A / S. Example 3 Human α₂MRAP production and folding human α₂-Macroglobulin receptor-related protein (α)₂Plasmid pT used for expression of MRAP) in E. coli BL21 cells₇H₆FX-α₂The conditions used for the production of MRAPs and fusion proteins were previously described by us in Nykjaer et al., J. Biol. Chem. 267: 14543-14546, 1992. The primers of SEQ ID NO: 9 and SEQ ID NO: 10 are α₂It was used in PCR, which is used to increase the DNA that encodes MRAP. MGSHHHHHHGSIEGR-α₂Crude protein extract precipitated from the phenolic phase of cell protein extracted from 2 liters of Escherichia coli BL21 cell culture expressing MRAP (where MGSHHHHHHGSIEGR is SEQ ID NO: 48), 6M guanidinium chloride and 50 mM Tris hydrochloride It was dissolved in a buffer containing dithioerythriol with a salt pH of 8 and 50 mM. After gel filtration into 8M urea, 0.5M sodium chloride, 50mM tris hydrochloride pH8 and 1mM methionine using Sephadex G-25 (Swedden Pharmacia), the fusion protein ie MGSHHHHHHGSIEGR-α₂For purification of MRAP (where MGSHHHHHHGSIEGR is SEQ ID NO: 48) (Hochuli et al., 1988), a crude protein preparation, Ni²⁺Activated by NTA-Agarose Matrix (Ni)²⁺It was applied to NTA-agarose) and subsequently moved to a periodic folding process. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Ni²⁺The preparation and "loading" of the NTA-agarose column is described in Example 1. Ni²⁺After applying the crude protein extract to the NTA-agarose column, the fusion protein or MGSHHHHHHGSIEGR-α₂MRAP (where MGSHHHHHHGSIEGR is SEQ ID NO: 48), 1 column volume of loading buffer, followed by 6 M guanidinium chloride, 50 mM Tris hydrochloride and 1 mM methionine, the absorbance (OD) of the eluate at 280 nm. Purified from most E. coli and λ phage proteins by washing until stable. The fusion protein was prepared using 0.5 M sodium chloride, 50 mM Tris hydrochloride pH 8, 2 mM calcium chloride and 1 mM 2-mercaptoethanol as buffer A, and 6 M, using a gradient management profile as described in Table 3. Guanidinium chloride, 50 mM Tris hydrochloride pH 8, 2 mM calcium chloride and 1 mM 2-mercaptoethanol as buffer B, Ni²⁺Regenerated on an NTA-agarose column. After the periodic folding process is completed, α₂MRAP fusion protein in Ni with a buffer containing 0.5 M sodium chloride, 50 mM Tris hydrochloride and 20 mM EDTA pH 8.²⁺Eluted from NTA-agarose column. Virtually Ni²⁺No agglutination or precipitation of the fusion protein was observed on the NTA-agarose column. α₂The evaluated yield of the MRAP fusion protein was 60 mg, and the efficiency of the folding process was close to 95%. Fusion protein MGSHHHHHHGSIEGR-α₂MRAP (where MGSHHHHHHGSIEGR is SEQ ID NO: 48) was cleaved by using bovine limiting protease FXa in elution buffer at a weight ratio of 200: 1 overnight at room temperature. After gel filtration with Sephadex G-25 into 100 mM sodium chloride and 25 mM Tris hydrochloride pH 8, the protein solution was Ni²⁺It was passed through an NTA-agarose column to remove the uncleaved fusion protein and the free fusion N-terminal tail derived from the cleaved fusion protein. Finally, dilute the protein solution 1: 4 with water, α₂The MRAP protein was purified from FXa by ion exchange chromatography using Q-Sepharose (Pharmacia, Sweden). The Q-Sepharose column was eluted with a 6-column volume linear gradient from 25 mM sodium chloride and 25 mM Tris hydrochloride pH 8 to 250 mM sodium chloride and 25 mM Tris hydrochloride pH 8. α₂The MRAP protein was eluted at the very beginning of the linear gradient. On the other hand, FXa was eluted later. Α produced and regenerated by this process₂The yield of MRAP protein was 40 mg. Ligand binding properties (ie α₂-Binding to macroglobulin receptors, and human urokinase plasminogen activator-plasminogen activator inhibitor type-I complex α₂-Interference with binding to the M receptor) was found to be identical to the ligand binding properties of the purified natural protein, according to Dr. Nykjaer. Example 4α₂-Domains and Domains from M Receptors-Production and Folding of Clusters Human α₂-Macroglobulin receptor / low density lipoprotein receptor related protein (α)₂MR) is a 600 kDa endocytosis membrane receptor. α₂-MR is synthesized as a 4524 amino acid single chain precursor protein. Its precursor is processed into an 85 kDa transmembrane β-chain and a 500 kDa α-chain that is non-covalently attached to the extracellular domain of the β-chain. α₂-MR is Ca²⁺(Ie, the reduced protein is Ca²⁺Is known to not combine), α₂-MR is believed to be multifunctional in the sense that it binds different classes of ligands. The entire amino acid sequence of the α-chain can be represented by three types of repeater clusters, which are also found in other membrane binding receptors and various plasma proteins. A: This type of repeater spans about 40 amino acid residues and is characterized by a sequential appearance of the six cysteine residues contained in the repeater. Some authors have named this repeat as a complement domain. B: This type of repeater is also characterized by the continuous appearance of the six cysteine residues contained in the repeater over about 40 amino acid residues. In the literature, this repeater is named the EGF-type domain. C: This type of repeater is characterized by the presence of a common sequence of SEQ ID NO: 39 over approximately 55 amino acid residues. This example is α₂-Several domains and domain clusters derived from MR proteins are produced in E. coli as fusion proteins cleavable by FXa, these recombinant proteins are purified, folded in vitro and cleaved by FXa. It states that it should be processed. Human α₂-A plasmid clone containing full-length cDNA encoding the MR protein (courtesy of Dr. Joachim Herz; Herz et al., EMBO J., 7: 4119-4127, 1988), α.₂-Used as a template in a series of polymerase chain reactions (PCRs) designed to produce cDNA fragments corresponding to several polypeptides representing domains and domain clusters derived from MR proteins. # 1 contains two domains of type A, α₂-Corresponds to amino acid residues 20-109 in MR proteins. The primers of SEQ ID NO: 11 and SEQ ID NO: 12 were used in PCR. # 2 contains two domains of type A followed by two domains of type B, α₂-Corresponds to amino acid residues 20-190 in MR proteins. The primers of SEQ ID NO: 11 and SEQ ID NO: 13 were used in PCR. # 3 consists of the same region as # 2 followed by a region containing the YWTD repeater, corresponding to amino acid residues 20-521. The primers of SEQ ID NO: 11 and SEQ ID NO: 14 were used in PCR. # 4 contains one domain of type B, followed by eight domains of type A, and finally two domains of type B, α₂-Corresponds to amino acid residues 803 to 1265 in MR proteins. The primers of SEQ ID NO: 15 and SEQ ID NO: 16 were used in PCR. # 5 contains only eight Type A domains that are also present in # 4, α₂-Corresponds to amino acid residues 849 to 1184 in MR proteins. The primers of SEQ ID NO: 17 and SEQ ID NO: 18 were used in PCR. # 6 contains two C-terminal type B domains from # 4 followed by eight YWTD repeaters and one type B domain, α₂-Corresponds to amino acid residues 1184 to 1582 in MR proteins. The primers of SEQ ID NO: 19 and SEQ ID NO: 20 were used in PCR. # 7 contains all the regions contained in the constructs from # 4 to # 6, α₂-Corresponds to amino acid residues 803 to 1582 in MR proteins. The primers of SEQ ID NO: 15 and SEQ ID NO: 20 were used in PCR. # 8 contains 10 type A domains, α₂-Corresponds to amino acid residues 2520 to 2941 in MR proteins. The primers of SEQ ID NO: 21 and SEQ ID NO: 22 were used in PCR. # 9 contains 11 type A domains, α₂-Corresponds to amino acid residues 3331 to 3778 in MR proteins. The primers of SEQ ID NO: 23 and SEQ ID NO: 24 were used in PCR. The amplified nucleotide sequence encoding these domains and domain clusters was subjected to PCR reaction at the 5'end of the amino acid sequence of SEQ ID NO: 37 (Nagai and Thφgersen, Methods in Enzymology, 152: 461-481,1987) was ligated into the encoding (included in SEQ ID NOs: 11, 15, 17, 19, 21 and 23) nucleotide sequences. E. coli expression vector pT₇H₆(Christensen et al., FEBS Letters, 295: 181-184, 1991), or pLcIIMLC (Nagai et al., Nature, 332) by inserting an oligonucleotide that encodes the six C-terminal histidine residues of the myosin light chain fragment. : 284-286, 1988) modified expression plasmid pLcIIMLCH₆The amplified DNA fragment was subcloned into one of the above. Obtained plasmid pT₇H₆FX- # 1 to # 3, and pLcIIMLCH₆The composition of FX- # 4 to # 9 is shown in Figures 6-8, and Figure 9 shows the amino acid sequence of the expressed protein (SEQ ID NO: 52 shows the amino acid sequence encoded by the full-length reading frame. Shows). pT₇H₆Domains and domain clusters subcloned into the FX series, on medium scale (2 liters), as described in Studier and Moffat, J. Mol.Biol., 189: 113-130,1986. Proliferated and expressed in E. coli BL21 cells. Cultures that grow exponentially at 37 ° C, OD₆₀₀ At 0.8, the bacteriophage λCE6 was infected with a multiplicity of infection of about 5. After growing the culture at 37 ° C for an additional 3 hours, cells were collected by centrifugation. Cells were lysed by osmotic shock and sonication and all cell proteins were extracted into phenol (adjusted to pH 8 with trizuma base). pLcIIMLCH₆Domain clusters subcloned into a series were grown and expressed in E. coli QY13 cells as described in Nagai and Thφgersen. Methods in Enzymology, 152: 461-481, 1987. Culture (4 liters) that grows exponentially at 30 ° C, OD₆₀₀ At 1.0, transferred to 42 ° C for 15 minutes. This heat shock induces the synthesis of fusion proteins. After incubating the culture at 37 ° C for an additional 3-4 hours, cells are collected by centrifugation. Cells were lysed by osmotic shock and sonication and all cellular proteins were extracted into phenol (adjusted to pH 8 with trizuma base). Crude protein was precipitated from the phenolic phase by the addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride and 50 mM tris hydrochloride pH 8 and 0.1 M dithioerythriol. Purification of fusion protein after gel filtration in 8M urea, 1M sodium chloride, 50mM tris hydrochloride pH8, 10mM 2-mercaptoethanol and 2mM methionine using Sephadex G-25 (Swedden Pharmacia). (Hochuli et al., 1988), the crude protein preparation, Ni²⁺The NTA-agarose column was applied to the activated NTA-agarose column, and the process was subsequently transferred to a periodic folding process. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Ni²⁺The preparation and "loading" of the NTA-agarose column is described in Example 1. Ni²⁺After applying the crude protein extract to the NTA-agarose column, the fusion protein was added to a loading buffer of 1 column volume, followed by 6M guanidinium chloride, 50 mM Tris hydrochloride, 10 mM 2-mercaptoethanol and 2 mM methionine. Purified from most E. coli and λ phage proteins by washing the eluate until its absorbance (OD) at 280 nm was stable. Reduction of each fusion protein to 0.5 M sodium chloride, 50 mM Tris hydrochloride pH 8, 2 mM calcium chloride, 0.33 mM methionine and 2.0 mM / 0.2 mM using a gradient management profile as shown in Table 4. Type / oxidized glutathione as buffer A, and 4M urea, 0.5M sodium chloride, 50 mM Tris hydrochloride pH 8, 2 mM calcium chloride, 2 mM methionine and 3 mM reduced glutathione as buffer B. , Ni²⁺The reduced / oxidized glutathione solution regenerated on the NTA-agarose column is 100-fold by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione prior to addition to buffer A. Newly prepared as a preservation solution. After the periodic folding process is completed, α₂-Fusion proteins representing domains and domain clusters derived from MR protein, using a buffer containing 0.5 M sodium chloride, 50 mM Tris hydrochloride and 5 mM EDTA pH 8 in Ni²⁺Eluted from NTA-agarose column. Ni²⁺The fusion protein that aggregated and precipitated on the NTA-agarose column was eluted in buffer B. α₂-Plasid pT representing the N-terminal 2 and 4 high cysteine domains of the MR protein₇H₆Approximately 75% of the fusion protein material expressed from FX- # 1 and # 2 is mixed with Ni by a non-denaturing buffer.²⁺It was eluted from the NTA-agarose column. Judging from non-reducing SDS-PAGE analysis, the majority of this fusion protein material appeared to be monomeric. The yield of monomeric fusion proteins # 1 and # 2 was assessed at approximately 50 mg. α₂-Approximately 50% of the fusion protein material expressed from all other expression plasmids, representing domain clusters derived from MR protein, in Ni with non-denaturing buffer.²⁺It was eluted from the NTA-agarose column. Judging from non-reducing SDS-PAGE analysis, 30% (fusion proteins # 5 and # 7) to 65% (fusion proteins # 4) of these fusion proteins appeared to be monomeric (Fig. 17). See lanes 9 and 10). Each fusion protein eluted with non-denaturing elution buffer was cleaved overnight at room temperature with an assessed weight ratio of 100: 1 using the limiting protease FXa. After gel filtration with Sephadex G-25 into 100 mM sodium chloride and 25 mM Tris hydrochloride pH 8, the protein solution was Ni²⁺It was passed through an NTA-agarose column to remove the uncleaved fusion protein and the free N-terminal fusion tail derived from the cleaved fusion protein. FXa was removed from the solution by passing the recombinant protein solution through a small column of SBTI-agarose {soy trypsin inhibitor immobilized on Sepharose CL-6B (Pharmacia, Sweden)}. SDS-PAGE analysis of the regenerated soluble fusion protein product # 4 is shown in lanes 9 and 10 of FIG. 17, which show reduced and non-reduced samples, respectively. The increased mobility seen in the non-reduced sample reflects the denseness of the polypeptide due to the presence of 33 disulfide bridges. Each recombinant protein is structurally Ca²⁺Was found to combine. Natural human α₂-The MR-derived monoclonal antibody A2MRα-5 was found by Dr. Sφren Moestrup to bind recombinant proteins expressed by constructs # 4, # 6 and # 7, while also natural α.₂-The unispecific antibody A2MRα-3 derived from MR was found to bind the recombinant protein expressed by construct # 8. The binding specificity of both antibodies is structure-dependent (ie, they are reduced α₂-Does not react with MR or recombinant protein). Example 5 Production and folding of bovine coagulation factor Xa (FXa) In this example, a fragment derived from bovine FXa is produced in E. coli as a fusion protein cleavable by FXa, and this recombinant protein is purified. , Described to be folded and processed in vitro. Amino acid residue Ser₈₂From Trp₄₈₄Up to (SEQ ID NO: 2, residues 82-484) bovine FX (FXΔ)_γThe first (1s) of the nucleotide sequence encoding the amino acid number (for the complete code reading frame) was synthesized from bovine whole liver RNA by the polymerase chain reaction (PCR).^tThe cDNA encoding bovine FX was cloned by specifically amplifying the cDNA using the oligo-dT of the strand as a primer as a template. The primers used in PCR were SEQ ID NO: 25 and SEQ ID NO: 26. RNA extraction and cDNA synthesis were performed using standard procedures. FXΔ_γAmino acid sequence of SEQ ID NO: 37 (Nagai and Thφgersen, Methods in Enzymology, 152: 461-481,) which constitutes the cleavage site of bovine restriction protease FXa by PCR reaction at the 5'end of the amplified reading frame encoding 1987) was linked to the encoding nucleotide sequence. E. coli expression vector pLcIIMLCH modified from pLcIIMLC (Nagai et al., Nature, 332: 284-286, 1988) by inserting an oligonucleotide that encodes the six C-terminal histidine residues of the myosin light chain fragment.₆The amplified DNA fragment was cloned into it. Obtained plasmid pLcIIMLCH₆FX-FXΔ_γThe composition of is shown in FIG. 10 and the amino acid sequence of the expressed protein is shown in FIG. 11 (SEQ ID NO: 53 shows the amino acid sequence encoded by the full-length reading frame). pLcIIMLCH₆-FXΔ_γThe plasmid was grown and expressed in E. coli QY13 cells as described in Nagai and Thφgersen (Methods in Enzymology, 152: 461-481, 1987). Cultures that grow exponentially at 30 ° C, OD₆₀₀ Incubated at 1.0 for 15 minutes at 42 ° C. This heat shock induces the synthesis of fusion proteins. After incubating the culture at 37 ° C for an additional 3-4 hours, cells are collected by centrifugation. Cells were lysed by osmotic shock and sonication and all cell proteins were extracted into phenol (adjusted to pH 8 with trizuma base). Crude protein was precipitated from the phenolic phase by the addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride and 50 mM tris hydrochloride pH 8 and 0.1 M dithioerythriol. Gel filtration using Sephadex G-25 (Pharmacia, Sweden, LKB) into 8M urea, 1M sodium chloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol, then FXΔ_γFor purification of fusion proteins (Hochuli et al., 1988), crude protein preparations, Ni²⁺It was applied to the NTA-agarose matrix activated by the above, and was subsequently transferred to a periodic folding process. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Ni²⁺The preparation and "loading" of the NTA-agarose column is described in Example 1. Ni²⁺After applying the crude protein extract to an NTA-agarose column, elute the fusion protein with 1 column volume of loading buffer, followed by 6M guanidinium chloride, 50 mM Tris hydrochloride and 10 mM 2-mercaptoethanol. Purified from most E. coli and λ phage proteins by washing until the absorbance (OD) at 280 nm was stable. This fusion protein was subjected to 0.5 M sodium chloride, 50 mM Tris hydrochloride pH 8, 2 mM calcium chloride and 2.0 mM / 0.2 mM reduced / oxidized glutathione using a gradient management profile as shown in Table 5. Ni as buffer A and with 8 M urea, 0.5 M sodium chloride, 50 mM Tris hydrochloride pH 8, 2 mM calcium chloride and 3 mM reduced glutathione as buffer B.²⁺Regenerated on an NTA-agarose column. The reduced / oxidized glutathione solution was newly prepared as a 100-fold storage solution by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione prior to addition to buffer A. FXΔ after the periodic folding process is completed_γThe fusion protein was added to Ni using a buffer containing 0.5 M sodium chloride, 50 mM Tris hydrochloride and 5 mM EDTA pH 8.²⁺Eluted from NTA-agarose column. Ni²⁺The fusion protein that aggregated and precipitated on the NTA-agarose column was eluted in buffer B. About 33% FXΔ_γFusion protein material is Ni with non-denaturing buffer²⁺Eluted from NTA-agarose column. FXΔ_γThe amount of fusion protein was assessed at about 15 mg. Judging from non-reducing SDS-PAGE analysis, only about one-third of this fusion protein material appeared to be monomeric. This corresponds to the overall efficiency of the folding process of about 10%. FXΔ in non-denatured buffer by passing the recombinant protein solution through a small column of trypsin-agarose {trypsin immobilized on Sepharose CL-6B (Pharmacia, Sweden)}_γActivated the fusion protein. Recombinant FXΔ activated using standard procedures utilizing chromogen substrates_γThe proteolytic activity and substrate specificity profile of the fusion protein was analyzed. The activity and substrate specificity profile were indistinguishable from those obtained from native bovine FXa. Example 6 Production and folding of kringle domains 1 and 4 from human plasminogen In this example, lysine-binding kringle domains 1 and 4 (K1 and K4, respectively) from human plasminogen can be cleaved by FXa. It describes that it is produced in Escherichia coli as a fusion protein, and that the K1- and K4-fusion proteins are purified and folded in vitro. A plasmid clone (courtesy of Dr. Earl Davie, Seattle, USA) containing a full-length cDNA that encodes human plasminogen cloned into the common cloning vector pUC18 was presented with K1 (so-called Glu-plasminogen). Amino acid residue Ser in₈₁From Glu₁₆₂(Equivalent to), and K4 (the amino acid residue Val in the so-called Glu-plasminogen)₃₅₄From Ala₄₃₉Used as a template in the polymerase chain reaction (PCR) designed to produce cDNA fragments corresponding to (corresponding to). The primers of SEQ ID NO: 27 and SEQ ID NO: 28 were used in the K1 producing PCR, and the primers of SEQ ID NO: 29 and SEQ ID NO: 30 were used in the K4 producing PCR. The amplified reading frame encoding K1 and K4 is contained in SEQ ID NO: 27 and SEQ ID NO: 29 by PCR reaction at the 5'end, and the amino acid sequence of SEQ ID NO: 37 constituting the cleavage site of the bovine restriction protease FXa. (Nagai and Thφgersen, Methods in Enzymology, 152: 461-481, 1987) were linked to the encoding nucleotide sequence. E. coli expression vector pLcIIMLCH modified from pLcIIMLC (Nagai et al., Nature, 332: 284-286, 1988) by inserting oligonucleotides encoding the six C-terminal histidinel residues of the myosin light chain fragment.₆The amplified K1 DNA fragment was cloned into it. Obtained plasmid pLcIIMLCH₆The configuration of FX-K1 is shown in Fig. 12. E. coli expression vector pLcIIH modified from pLcII (Nagai and Thφgersen. Methods in Enzymology, 152: 461-481, 1987) by inserting oligonucleotides encoding the six C-terminal histidinel residues of the cII fragment.₆The amplified K4 DNA fragment was cloned into it. Obtained plasmid pLcIIH₆The composition of FX-K4 is shown in FIG. 13, and the amino acid sequence of human "Glu" -plasminogen (SEQ ID NO: 54) is shown in FIG. pLcIIMLCH₆-K1 plasmid and pLcIIH₆Both FX-K4 plasmids were grown and expressed in E. coli QY13 cells as described in Nagai and Thφgersen. Methods in Enzymology, 152: 461-481, 1987. Cultures that grow exponentially at 30 ° C, OD₆₀₀ At 1.0, transferred to 42 ° C for 15 minutes. This heat shock induces the synthesis of fusion proteins. After incubating the culture at 37 ° C for an additional 3-4 hours, cells are collected by centrifugation. Cells were lysed by osmotic shock and sonication and all cell proteins were extracted into phenol (adjusted to pH 8 with trizuma base). The crude protein was precipitated from the phenol phase by adding 2.5 volumes of ethanol and centrifuging. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride and 50 mM tris hydrochloride pH 8 and 0.1 M dithioerythriol. Gel filtration into 8 M urea, 1 M sodium chloride, 50 mM Tris hydrochloride pH 8, 10 mM 2-mercaptoethanol and 2 mM methionine using Sephadex G-25 (Pharmacia, Sweden), then K1- and K4 -For purification of fusion proteins (Hochuli et al., 1988), crude protein preparations, Ni²⁺It was applied to the NTA-agarose matrix activated by the above, and was subsequently transferred to a periodic folding process. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Ni²⁺The preparation and "loading" of the NTA-agarose column is described in Example 1. Ni²⁺After applying the crude protein extract to the NTA-agarose column, the fusion protein was added to a loading buffer of 1 column volume, followed by 6M guanidinium chloride, 50 mM Tris hydrochloride, 10 mM 2-mercaptoethanol and 2 mM methionine. Purified from most E. coli and λ phage proteins by washing the column eluate until the absorbance (OD) at 280 nm was stable. This fusion protein was subjected to 0.5 M sodium chloride, 50 mM Tris hydrochloride pH 8, 10 mM 6-aminohexanoic acid (ε-aminocaproic acid, ε-ACA), 0.33, using a gradient management profile as shown in Table 4. With mM methionine and 2.0 mM / 0.2 mM reduced / oxidized glutathione as buffer A, and 4 M urea, 0.5 M sodium chloride, 50 mM Tris hydrochloride pH 8, 10 mM ε-ACA, 2 mM methionine and Ni using 3 mM reduced glutathione as buffer B²⁺Regenerated on an NTA-agarose column. The reduced / oxidized glutathione solution was newly prepared as a 100-fold storage solution by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione prior to addition to buffer A. After the periodic folding process is complete, each of the K1- and K4-fusion proteins is added to Ni with a buffer containing 0.5 M sodium chloride, 50 mM Tris hydrochloride and 5 mM EDTA pH 8.²⁺Eluted from NTA-agarose column. Ni²⁺The fusion protein that aggregated and precipitated on the NTA-agarose column was eluted in buffer B. Virtually all K1- and K4-fusion protein materials are Ni with non-denaturing buffer.²⁺Eluted from NTA-agarose column. The evaluated yields of K1-fusion protein and K4-fusion protein were about 60 mg. Judging from non-reducing SDS-PAGE analysis, virtually all K1-fusion and K4-fusion proteins appeared to be monomeric. This corresponds to a folding process efficiency of rubbing 90%. SDS-PAGE analysis of the production of recombinant plasminogen kringle 1 and 4 is shown in FIG. In addition, the K1-fusion protein and K4-fusion protein were purified by affinity chromatography with lysine-cepharose CL-6B (Pharmacia, Sweden). The fusion protein was eluted from the affinity column using a buffer containing 0.5 M sodium chloride and 50 mM Tris hydrochloride pH 8 and 10 mM ε-ACA. Binding to lysine-cepharose is usually taken as an indicator of correct folding of the lysine-binding Kringle domain. The three-dimensional structure of the recombinant K1 and K4 protein domains produced by this periodic folding step, completely processed by liberation from the N-terminal fusion tail, and subsequently purified using ion exchange chromatography is X. Confirmed using X-ray diffraction (performed by Dr. Robert Huber) and 2D NMR analysis {performed by stud.scient. Peter Reinholdt and Dr. Flemming Poulsen}. The approximate yield of recombinant K1 and K4 protein domains fully processed by this step is 5 mg per liter of culture. Example 7 Human α₂-Production and regeneration of recombinant fragments derived from macroglobulins and chicken ovostatin in E. coli This example is a human α as an FXa cleavable fusion protein.₂-Macroglobulin (α₂-Production of receptor-binding domain of MRBDv) in E. coli, and recombinant α after FXa cleavage₂-Describes purification of MRBDv. Amino acid residue Val₁₂₉₉From Ala₁₄₅₁Up to α₂-Macroglobulin reading frame (α)₂-462 base pair (bp) DNA fragments encoding MRDv) were amplified by the polymerase chain reaction (PCR) method substantially according to the protocol of Saiki et al. (1988). Human α₂-PA2M (kindly provided by Dr. T. Kristensen) containing the full-length cDNA of macroglobulin was used as a template, and the oligonucleotides of SEQ ID NO: 31 and SEQ ID NO: 32 were used as primers. The amplified code reading frame encodes the amino acid sequence of SEQ ID NO: 37 (Nagai and Thφgersen, 1987) contained in SEQ ID NO: 7 and constituting the cleavage site of bovine limiting protease FXa by PCR reaction at the 5'end. It was linked to a nucleotide sequence. Amplified DNA fragment is expressed in E. coli expression vector pT₇H₆Subcloned into (Christensen et al., 1991). Obtained plasmid pT₇H₆FX-α₂-MRDv (Human α)₂The composition of -MRDv (expressing) is shown in FIG. 18, and the amino acid sequence of the expressed protein is shown in FIG. 19 (SEQ ID NO: 55). Recombinant human α₂MRDv, plasmid pT in E. coli BL21 cells on medium scale (2 x 1 liter) as described in Studier and Moffat, J. Mol.Biol., 189: 113-130,1986.₇H₆FX-α₂It was produced by growing and expressing MRDv. Cultures that grow exponentially at 37 ° C, OD₆₀₀ At 0.8, the bacteriophage λCE6 was infected with a multiplicity of infection of about 5. After growing the culture at 37 ° C for an additional 3 hours, cells were collected by centrifugation. Cells were lysed by osmotic shock and sonication and all cellular proteins were extracted into phenol (adjusted to pH 8 with trizuma base). Protein was precipitated from the phenolic phase by the 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 hydrochloride pH 8 and 50 mM dithioerythriol. After gel filtration with Sephadex G-25 (Sweden Pharmacia, LKB) into 8M urea, 1M sodium chloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol, the fusion protein, ie MGSHHHHHHGSIEGR- α₂For purification of MRDv (where MGSHHHHHHGSIEGR is SEQ ID NO: 48) (Hochuli et al., 1988), a crude protein preparation, Ni²⁺Activated by NTA-Agarose Column (Ni)²⁺It was applied to NTA-agarose) and subsequently moved to a periodic folding process. Ni²⁺The preparation and "loading" of the NTA-agarose column is described in Example 1. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Ni²⁺After applying the crude protein extract to the NTA-agarose column, the fusion protein, ie MGSHHHHHHGSIEGR-α₂MRDv (where MGSHHHHHHGSIEGR is SEQ ID NO: 48), 1 column volume of loading buffer, followed by 6 M guanidiinium chloride, 50 mM Tris hydrochloride and 10 mM 2-mercaptoethanol, the absorbance of the eluate at 280 nm. Purified from most E. coli and λ phage proteins by washing until (OD) was stable. The fusion protein was prepared using 0.5 M sodium chloride and 50 mM Tris hydrochloride pH 8 and 2.0 mM / 0.2 mM reduced / oxidized glutathione as buffer A, using a gradient management profile as described in Table 4. Ni with 8M urea, 0.5M sodium chloride, 50 mM Tris hydrochloride pH 8 and 5 mM reduced glutathione as buffer B.²⁺Regenerated on an NTA-agarose column. The reduced / oxidized glutathione solution was newly prepared as a 200-fold storage solution by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione prior to addition to buffer A. After the periodic folding process is completed, α₂MRDv fusion protein in Ni with a buffer containing 0.5 M sodium chloride, 50 mM Tris hydrochloride and 20 mM EDTA pH 8.²⁺Eluted from NTA-agarose column. Ni²⁺The fusion protein that aggregated and precipitated on the NTA-agarose column was eluted in buffer B. Approximately 50% of the fusion protein material was eluted with aqueous elution buffer. Judging from non-reducing SDS-PAGE analysis, half of this fusion protein material appeared to be monomeric and folded. Recombinant α₂The MRDv protein was released from the N-terminal fusion tail by cleaving at room temperature for 4 hours at a weight ratio of approximately 50: 1 with the limiting protease FXa. After cutting, α₂The MRDv protein was combined with the uncleaved fusion protein by gel filtration with Sephadex G-25 into 10 mM sodium chloride and 50 mM Tris hydrochloride pH 8 and subsequently by ion exchange chromatography with Q-Sepharose. Isolated from free fusion tail and FXa. α₂MRDv was eluted with a linear gradient (over 10 column volumes) from 10 mM sodium chloride and 10 mM Tris hydrochloride pH 8 to 500 mM sodium chloride and 10 mM Tris hydrochloride pH 8. α₂The MRDv protein was eluted with 150 mM sodium chloride. Recombinant α₂MRDv domain is α₂Complete α to M-receptor₂-K in {1 ligand-1 receptor binding (Moestrup and Gliemann 1991) that binds with the same affinity that the macroglobulin molecule shows for its receptor_DRegarding evaluation}. The binding analysis was performed by Dr. Sφren K. Moestrup and a scientific research student (stud. Scient.) Kare Lehmann. Example 8 Production and regeneration of recombinant fragment derived from Nijimas virus VHS envelope glycoprotein G in Escherichia coli Expression of recombinant fragment derived from envelope glycoprotein G from Nijimas virus VHS in Escherichia coli as an FXa cleavable fusion protein In vitro (in vitro) regeneration was performed using a general strategy and method similar to that set forth in the general description of "Periodic Regeneration Steps" and listed in Examples 1-6. Example 9 Production and regeneration of recombinant human tetranectin and recombinant fragments in E. coli, derived from human Tetranectin, are four identical and non-covalent bindings of 181 amino acid residues (17 kDa). It is a tetrameric protein consisting of single-chain subunits linked to. Each subunit has 3 disulfide bridges and Ca²⁺To combine. Tetranectin is found in plasma and associates with the extracellular matrix. Tetranectin specifically binds to plasminogen kringle 4. This bond can be specifically titrated with lysine or ω-amino acid. Amino acid residue Glu₁From Val₁₈₁Nucleotide sequences up to (1s) were synthesized from human whole placental RNA by the polymerase chain reaction (PCR) (Saiki et al., 1988).^t) The cDNA encoding the reading frame corresponding to the mature tetranectin single-stranded subunit was cloned by specifically amplifying the cDNA with the oligo-dT of the strand as a primer as a template. The primers used in 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 monomeric subunit of tetranectin was subjected to PCR reaction at the 5'end to form the amino acid sequence of SEQ ID NO: 37 (Nagai, and Thφgersen, 1987) that constitutes the cleavage site of the bovine limiting protease FXa. Was linked to the nucleotide sequence that encodes. Due to the specific design of the 5'-PCR primer (SEQ ID NO: 33), glycine residues were added to the C-terminal arginine residue at the FXa cleavage site (SEQ ID NO: 37) and tetranectin Glu.₁-Insert between residues. Amplified DNA fragment is expressed in E. coli expression vector pT₇H₆Subcloned into (Christensen et al., 1991). Obtained plasmid pT₇H₆The composition of FX-TETN (expressing a tetranectin monomer) is shown in FIG. 20, and the amino acid sequence of the expressed protein is shown in Figure 21 (SEQ ID NO: 56, encoded by a full-length reading frame). The amino acid sequence is shown). Plasmid pT to prepare tetranectin monomer₇H₆FX-TETN, medium scale (4 x 1 liter; 2 x TY medium, 5 mM magnesium sulphate and 100 μg, as described in Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986. Ampicillin) was grown in Escherichia coli BL21 cells. Cultures that grow exponentially at 37 ° C, OD₆₀₀ At 0.8, the bacteriophage λCE6 was infected with a multiplicity of infection of about 5. Cultures were grown at 37 ° C for an additional 3 hours and cells were collected by centrifugation. Cells were resuspended in 150 ml of 0.5 M sodium chloride, 10 mM Tris hydrochloride pH 8 and 1 mM EDTA pH 8. Phenol (100 ml adjusted to pH 8) was added and the mixture was sonicated to extract all proteins. 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 hydrochloride pH 8 and 0.1 M dithioerythriol. After gel filtration with Sephadex G-25 (Sweden Pharmacia, LKB) into 8M urea, 1M sodium chloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol, the fusion protein, ie MGSHHHHHHGSIEGR- For purification of TETN (where MGSHHHHHHGSIEGR is SEQ ID NO: 48) (Hochuli et al., 1988), a crude protein preparation, Ni²⁺Activated by NTA-Agarose Column (Ni)²⁺Applied to NTA-agarose, 8M urea, 1M sodium chloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol (75 ml) pre-washed. Ni²⁺The preparation and "loading" of the NTA-agarose column is described in Example 1. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Columns were 8M urea, 1M sodium chloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol 200 ml (buffer I), and 6 M guanidinium chloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-. It was washed with 100 ml of mercaptoethanol (buffer II). The MGSHHHHHHGSIEGR-TETN fusion protein was eluted with buffer II containing 10 mM EDTA pH 8, and the eluate was gel filtered through Sephadex G25 using buffer I as the eluent. Next, the eluted protein was regenerated. 100 ml of Ni on the fusion protein MGSHHHHHHGSIEGR-TETN (where MGSHHHHHGSIEGR is SEQ ID NO: 48)²⁺NTA-agarose was mixed. The resin containing the bound protein was packed in a column having a diameter of 5 cm and washed with buffer I supplemented with calcium chloride up to 2 mM. The fusion protein buffers 0.5 M sodium chloride, 50 mM Tris hydrochloride pH 8, 2 mM calcium chloride and 2.0 mM / 0.2 mM reduced / oxidized glutathione using a gradient management profile as shown in Table 4. Ni as buffer A and with 8M urea, 1M sodium chloride, 50 mM Tris hydrochloride pH 8, 2 mM calcium chloride and 3 mM reduced glutathione as buffer B.²⁺Regenerated at 11-12 ° C on an NTA-agarose column. The reduced / oxidized glutathione solution was newly prepared as a 200-fold storage solution by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione prior to addition to buffer A. After the periodic folding process is complete, the tetranectin fusion protein is added to Ni with a buffer containing 0.5 M sodium chloride, 50 mM Tris hydrochloride and 25 mM EDTA pH 8.²⁺Eluted from NTA-agarose column. The tetranectin fusion protein was cleaved with FXa at a molar ratio of 1: 300 at 4 ° C. overnight. After FXa cleavage, the protein sample was concentrated 10-fold by ultrafiltration using a YM10 membrane (Amicon). After diluting the protein sample 10-fold with 2 mM calcium chloride, 10 mM tris hydrochloride pH 8 and 2 mM calcium chloride to 10 mM tris hydrochloride pH 8 and 2 mM by ion exchange chromatography with Q-Sepharose (Sueden's Pharmacia). Recombinant tetranectin was isolated in a linear gradient of calcium chloride and 0.5 M sodium chloride over 10 column volumes. Recombinant tetranectin produced by this step was analyzed by Dr. Inge Clemmensen Rigshospitalet of Copenhagen. Dr. Clemmensen found that recombinant tetranectin behaved similarly to naturally isolated human tetranectin with respect to binding to plasminogen kringle 4 and expression of antigenic sites. Ni using "periodic regeneration process"²⁺A preliminary experiment comparing the regeneration efficiency of the recombinant tetranectin fusion protein bound to the NTA-agarose column with the recombinant tetranectin in the dialysis bag showed a significant yield of soluble monomers from the solution regeneration strategy. It clearly shows that it has been improved. However, if any product of the circulation step is disulfide re-shuffled in a solution in the presence of 5 mM calcium chloride, substantially all polypeptide materials will be properly folded tetranectin. Converted to a tetramer. Denatured and reduced recombinant tetranectin in a dialysis bag, buffer B (6M urea, 100 mM sodium chloride, 50 mM tris hydrochloride pH = 8, 2 mM / 0.2 mM reduced form / Oxidized glutathione, 2 mM calcium chloride and 0.5 mM methionine) and buffer A (100 mM sodium chloride, 50 mM tris hydrochloride pH 8, 2 mM / 0.2 mM reduced / oxidized glutathione, 2 mM calcium chloride and 0 It was regenerated by exposure to (5 mM methionine) over 15 cycles. Example 10 Production and folding of intracellularly expressed diabody in E. coli: Mab32 diabody diabody specific for tumor necrosis factor (described in Hollinger et al., 1993) is an artificial divalent antibody. It is a highly specific antibody fragment. In this example, diabodies from the mouse monoclonal antibody Mab32 (Rathjen et al., 1991, 1992; Australian patent application 7,576; EP-A-486,526) specific for tumor necrosis factor alpha (TNF-α). The production of in E. coli is described. The phagemid clone pCANTAB5-myc-Mab32-5 containing Mab32 encoded in the diabodies format (PCT / GB93 / 02492) is available from Cambridge Antibody Technology (CAT) Ltd., Cambridge, UK G.M. Also generously provided by Dr. Winter. pCANTAB5-myc-Mab32-5DNA is a polymerase chain reaction (PCR) (PCR) (Saiki et al.) Designed to produce cDNA fragments corresponding to artificial complete diabodies using primers of SEQ ID NO: 35 and SEQ ID NO: 36. It was used as a template in 1988). Nucleotides contained in SEQ ID NO: 35 encoding the amino acid sequence of SEQ ID NO: 37 constituting the cleavage site of bovine limiting protease FXa (Nagai and Thogersen, 1987) through a PCR reaction with an amplification code reading frame at the 5'end. It was concatenated into an array. 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 (expressing Mab32 diabodies) is shown in FIG. 22. The amino acid sequence of the expressed protein is shown in FIG. 23 (SEQ ID NO: 57 shows the amino acid sequence encoded by the full-length reading frame). To generate the diabodies, the plasmid pT7H6FX-DB32 was placed on a medium scale in E. coli BL21 cells as described in Studier and Moffat, J. Mol.Biol., 189: 113-130, 1986. It was grown in 4 x 1 liter; 2 x TY medium, 5 mM pyridine 4 and 100 μg ampicillin). Cultures that grow exponentially at 37 ° C, at a multiplicity of about 5, 0D It was used as a template in 1988). Nucleotides contained in SEQ ID NO: 35 encoding the amino acid sequence of SEQ ID NO: 37 constituting the cleavage site of bovine limiting protease FXa (Nagai and Thogersen, 1987) through a PCR reaction with an amplification code reading frame at the 5'end. It was concatenated into an array. 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 (expressing Mab32 diabodies) is shown in FIG. 22. The amino acid sequence of the expressed protein is shown in FIG. 23 (SEQ ID NO: 57 shows the amino acid sequence encoded by the full-length reading frame). To generate the diabodies, the plasmid pT7H6FX-DB32 was placed on a medium scale in E. coli BL21 cells as described in Studier and Moffat, J. Mol.Biol., 189: 113-130, 1986. It was grown in 4 x 1 liter; 2 x TY medium, 5 mM pyridine 4 and 100 μg ampicillin). Cultures that grow exponentially at 37 ° C, at a multiplicity of about 5, 0D It was used as a template in 1988). Nucleotides contained in SEQ ID NO: 35 encoding the amino acid sequence of SEQ ID NO: 37 constituting the cleavage site of bovine limiting protease FXa (Nagai and Thogersen, 1987) through a PCR reaction with an amplification code reading frame at the 5'end. It was concatenated into an array. 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 (expressing Mab32 diabodies) is shown in FIG. 22. The amino acid sequence of the expressed protein is shown in FIG. 23 (SEQ ID NO: 57 shows the amino acid sequence encoded by the full-length reading frame). To generate the diabodies, the plasmid pT7H6FX-DB32 was placed on a medium scale in E. coli BL21 cells as described in Studier and Moffat, J. Mol.Biol., 189: 113-130, 1986. It was grown in 4 x 1 liter; 2 x TY medium, 5 mM pyridine 4 and 100 μg ampicillin). Cultures that grow exponentially at 37 ° C, at a multiplicity of about 5, 0D , 1991). The construction of the resulting plasmid pT7H6FX-DB32 (expressing Mab32 diabodies) is shown in FIG. 22. The amino acid sequence of the expressed protein is shown in FIG. 23 (SEQ ID NO: 57 shows the amino acid sequence encoded by the full-length reading frame). To generate the diabodies, the plasmid pT7H6FX-DB32 was placed on a medium scale in E. coli BL21 cells as described in Studier and Moffat, J. Mol.Biol., 189: 113-130, 1986. It was grown in 4 x 1 liter; 2 x TY medium, 5 mM pyridine 4 and 100 μg ampicillin). Cultures that grow exponentially at 37 ° C, at a multiplicity of about 5, 0D , 1991). The construction of the resulting plasmid pT7H6FX-DB32 (expressing Mab32 diabodies) is shown in FIG. 22. The amino acid sequence of the expressed protein is shown in FIG. 23 (SEQ ID NO: 57 shows the amino acid sequence encoded by the full-length reading frame). To generate the diabodies, the plasmid pT7H6FX-DB32 was placed on a medium scale in E. coli BL21 cells as described in Studier and Moffat, J. Mol.Biol., 189: 113-130, 1986. It was grown in 4 x 1 liter; 2 x TY medium, 5 mM pyridine 4 and 100 μg ampicillin). Cultures that grow exponentially at 37 ° C, at a multiplicity of about 5, 0D₆₀₀ At 0.8, it was infected with bacteriophage λCE6. 40 minutes after infection, rifampicin was added (0.2 g in 2 ml methanol per liter of medium). Cultures were grown at 37 ° C. for an additional 3 hours and cells were collected by centrifugation. Cells were resuspended in 150 ml of 0.5 M NaCl, 10 mM Tris hydrochloride pH 8, and 1 mM EDTA pH 8. Phenol (100 ml, adjusted to pH 8) was added and the mixture was sonicated to extract total protein. The protein was precipitated from the phenol phase by centrifugation with 2.5 volumes of ethanol. The protein pellet was dissolved in buffer containing 6 M guanidinium chloride, 50 mM tris hydrochloride pH 8 and 0.1 M dithioerythriol. After gel filtration into 8M urea, 1M NaCl, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol using Sephadex G-25 (Pharmacia, LKB, Sweden), the crude protein sample is fused protein. For purification of MGSHHHHHHGSIEGR-DB32 (here MGSHHHHHGSIEGR is SEQ ID NO: 48), Ni²⁺Activated NTA-agarose column (8M urea, 1M NaCl, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol pre-washed Ni²⁺Applied to NTA-agarose, 75 ml). The preparation and "loading" of the Ni2 + NTA-agarose column is described in Example 1. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Columns are 8M urea, 1M NaCl, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol (buffer I) 200 ml, and 6M guanidinium chloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol ( It was washed with 100 ml of buffer II). The MGSHHHHHHGSIEGR-DB32 fusion protein was eluted with buffer II containing 10 mM EDTA pH 8, and this eluate was gel filtered through Sephadex G25 using buffer I as the eluent. The eluted protein was then regenerated. Add 100 ml of the fusion protein MGSHHHHHHGSIEGR-DB32 (here MGSHHHHHGSIEGR is SEQ ID NO: 48) to 100 ml of Ni.²⁺Mixed with NTA-agarose. The resin containing the binding protein was packed in a column having a diameter of 5 cm and washed with buffer I. Fusion proteins were presented in the gradient management profile shown in Table 4, with 0.5 M NaCl as buffer A, 50 mM Tris hydrochloride pH 8 and 2.0 mM / 0.2 mM reduced / oxidized glutathione, and 8 M urea as buffer B. , 1M NaCl, 50 mM Tris hydrochloride pH 8 and 3 mM reduced glutathione, Ni at 11-12 ° C.²⁺Regenerated on an NTA-agarose column. The reduced / oxidized glutathione solution was newly prepared as a 200-fold storage solution by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione before adding to buffer A. After completion of the periodic folding process, Ni was added to the DB32 fusion protein with a buffer containing 0.5 M NaC, 50 mM Tris hydrochloride and 25 mM EDTA pH 8 and adjusted to 5 mM GSH, 0.5 mM GSSG.²⁺It was eluted from the NTA-agarose column and incubated at 20 ° C for 12-15 hours. The fusion protein was then concentrated 50-fold by ultrafiltration using a YM10 membrane and purified by centrifugation. The DB32 fusion protein dimer was purified by gel filtration using a Superrose 12 column (Pharmacia, Sweden) using PBS as the eluent. The total yield of properly folded DB32 fusion protein obtained by this step was 4 mg per liter. Analysis by non-reducing SDS-PAGE from different stages of purification is shown in Figure 26. The MGSHHHHHHGSIEGR (SEQ ID NO: 48) N-terminal fusion peptide was cleaved from the DB32 protein at 37 ° C for 20 hours by cleavage with the limiting protease FXa (molar ratio 1: 5 FXa: DB32 fusion protein). This is shown in FIG. 26 as the appearance of a lower molecular weight band just below the uncleaved fusion protein. The regenerated DB32 protein is Cambridge Antibody Techonology Analyzed by Ltd. (CAT). DB32 was found to specifically bind to TNF-α and compete with the entire Mab32 antibody for binding to TNF-α. In addition, both DB32 and Mab32 compete for binding to TNF-α by sheep anti-301 antisera. Sheep anti-301 antisera are produced by immunizing sheep with a peptide that encodes the first 18 amino acids of human TNF-α and contains at least some of the epitopes recognized by mouse Mab32. Example 11 Production and regeneration of human saliasin in E. coli Saliasin is a single-domain Ca consisting of 100 amino acid residues.²⁺-It is a binding protein. Salaicin contains a single disulfide bridge. Proteins believed to belong to the S100 protein group are highly regulated in psoriatic skin and primary keratin cells in humans undergoing unusual differentiation. Plasmid pT₇H₆FX-PS.4 (kindly brought by Dr. P. Madsen of the Insitute of Medical Biochemistry, University of Aarhus, Demkark) was previously described by Hoffmann et al. (1994). Ser₂From Gln₁₀₁The nucleotide sequence encoding the saliacin protein up to is linked at the 5'end to the nucleotide sequence encoding the amino acid sequence MGSHHHHHHGSIEGR (SEQ ID NO: 48). pT₇H₆The map of FX-PS.4 is shown in Figure 24, and the amino acid sequence of human saliacin is shown in Figure 25 (SEQ ID NO: 58 shows the amino acid sequence encoded by the full-length reading frame. It is shown). Recombinant human saliacin, plasmid pT in E. coli BL21 cells₇H₆It was proliferated and expressed from FX-PS.4, and whole cell proteins were extracted as described (Hoffmann et al., 1994). The total ethanol-precipitated protein was dissolved in buffer containing 6 M guanidinium chloride, 50 mM Tris hydrochloride pH 8 and 50 mM dithioerythriol. After gel filtration into 8M urea, 0.5M NaCl, 50mM tris hydrochloride pH 8 and 5mM 2-mercaptoethanol using Sephadex G-25 (Pharmacia, LKB, Sweden), the crude protein sample was subjected to a fusion protein. MGSHHHHHHGSIEGR-For purification of saliacin (here MGSHHHHHGSIEGR is SEQ ID NO: 48) (Hochuli et al., 1988), Ni²⁺Activated NTA-Agarose Column (Ni²⁺It was applied to NTA-agarose), followed by a periodic folding process. Ni²⁺The preparation and "loading" of the NTA-agarose column is described in Example 1. All buffers prepared for liquid chromatography were degassed under vacuum prior to the addition and / or use of the reducing agent. Ni²⁺After applying the crude protein extract to the NTA-agarose column, add the fusion protein MGSHHHHHHGSIEGR-salaicin (here MGSHHHHHGSIEGR is SEQ ID NO: 48) in a single column amount until the 280 nm absorbance (OD) of the eluate stabilizes. Purification was performed from the majority of E. coli and λ phage proteins by washing with a loading buffer followed by 6M guanidinium chloride, 50 mM Tris hydrochloride and 5 mM 2-mercaptoethanol. The fusion proteins were presented in the gradient management profile shown in Table 4, 0.5 M NaCl as buffer A, 50 mM Tris hydrochloride pH 8, 2 mM CaCl2 and 1.0 mM / 0.1 mM reduced / oxidized glutathione, buffer B. As 8M urea, 0.5M NaCl, 50 mM Tris hydrochloride pH8, 2 mM CaCl₂And with 5 mM reduced glutathione, Ni²⁺Regenerated on an NTA-agarose column. The reduced / oxidized glutathione solution was newly prepared as a 200-fold storage solution by adding 9.9 M hydrogen peroxide to a stirred solution of 0.2 M reduced glutathione before adding to buffer A. After completion of the periodic folding procedure, Ni is a buffer containing saliacin fusion protein containing 0.5 M NaCl, 50 mM Tris hydrochloride, and 10 mM EDTA pH 8.²⁺Eluted from NTA-agarose column. Ni²⁺The fusion protein aggregated and precipitated on the NTA-agarose column was eluted in buffer B. Approximately 95% of the fusion protein material was eluted with non-denaturing elution buffer. Judging by non-reducing SDS-PAGE analysis, 75% of this soluble fusion protein material appeared to be monomeric, and the overall efficiency of the folding process was about 70%. Production of recombinant human saliacin (Hoffman et The efficiency of the above-mentioned regeneration process with respect to al., 1994) was evaluated to be less than 25%. The saliacin fusion protein was cleaved with FXa at a molar ratio of 100: 1 at room temperature for 48 hours. After gel filtration using Sephadex G-25 in a buffer containing 20 mM sodium acetate pH 5 and 20 mM NaCl, the protein sample was applied to an S-Sepharose ion exchange column (Pharmacia). The monomeric recombinant saliacin was eluted over 5 column volumes by a linear gradient from 20 mM sodium acetate pH 8 and 20 mM NaCl to 0.5 M NaCl. The monomeric saliacin was eluted with 150 mM NaCl. The saliacin dimer and higher multimers, along with the uncleavable fusion protein, eluted later in the gradient. Fractions containing the cleaved purified recombinant protein were gel filtered using Sephadex G25 into a buffer containing 150 mM NaCl, 10 mM Tris hydrochloride pH 7.4 and stored at 4 ° C. Example 12 Determination of Optimal Levels for Modulators and Disulfide Reorganizers for Maximizing Use of Thiol Compounds as Reducing Agents in Periodic Regeneration Procedures and Periodic Regeneration Steps Periodic Regeneration In order to increase the yield of properly folded protein obtained from, the number of production cycles should be maximized (see Overview of the Invention). In the production cycle, during the regeneration process of that cycle, misfolded proteins are rescued into an unfolded conformation on the way to a dead-end agglutinating conformation, while most of the already properly folded proteins are regenerated. It is characterized by a process of denaturation that remains in a conformational state that can bounce back to its original state. Proteins containing some disulfide bridges are β₂-It is known to regenerate with high efficiency (&gt; 95%) when exposed to the highest levels of denaturing agents as long as the disulfide bridge is intact, such as microglobulin. In this example, a method of assessing the suitability of a thiol compound for periodic regeneration based on its ability to distinguish between correct and incorrect disulfide bridges, and the level of denaturing and / or reducing agents used in the denaturing step is produced. The method of optimizing to maximize the number of cycles is described. Purified recombinant human β as a model system₂-Choose a mixture of mono, di, and multimeric forms of microglobulin. Its specific purpose is to have different topological forms of human β for reduction by five different reducing agents when the concentration of the denaturing agent is changed.₂-It was to analyze the stability of microglobulins. Human β in 6M guanidinium chloride, 50 mM Tris hydrochloride and 10 mM 2-mercaptoethanol pH 8₂-Microglobulin (produced as described in Example 13), non-denatured buffer (50 mM Tris hydrochloride, 0.5 M NaCl) Gel filtration was performed during pH 8). Only the protein fraction in the sample was dissolved in non-denaturing buffer. After exposure to air for 48 hours, the protein solution became opaque. Non-reducing SDS-PAGE analysis revealed that most proteins were oxidized to multimeric forms, and only small fractions were oxidized to be monomeric (Figure 27, Lane 1) with some protein solutions. The tubes were aliquoted and added in varying amounts of urea, keeping the protein and salt concentrations at constant levels. A reducing agent of either glutathione, cystine ethyl ester, N-acetyl-L-cystine, mercaptosuccinic acid or 2-mercaptoethanol was added to a collection of protein samples with varying urea concentrations. Each reducing agent was added to a final concentration of 4 mM. The protein sample was incubated at room temperature for 10 minutes, after which the free thiol groups were blocked by adding iodoacetic acid to a final concentration of 12 mM. Finally, protein samples were analyzed using non-reducing SDS-PAGE (Fig. 27-32). The composition of the test samples used for non-reducing SDS-PAGE is shown in the table below, as well as the results. In the line representing the ability of the reducing agent chosen to reduce the disulfide bridge, the "+++" mark represents a good ability, the "++" represents an intermediate ability, and the "+" indicates a weak ability. Those without a one-sided mark indicate that no measurable effect was observed.<img file="JP3695467B2_D0008.tif" /><img file="JP3695467B2_D0009.tif" /><img file="JP3695467B2_D0010.tif" /><img file="JP3695467B2_D0011.tif" /><img file="JP3695467B2_D0012.tif" />Beta of different topology form₂-m can be separated by non-reducing SDS-PAGE gel electrophoresis. The band that migrates fastest represents the oxidized monomer type. Immediately after this band, reduced β with a slightly slower migration rate₂-m follows, while the multimeric form of the protein is running much slower in the gel. In this analysis, β without significantly reducing the correctly formed disulfide bridges in the oxidized form of the monomer.₂-We are verifying the ability of each of the five tested reducing agents to reduce disulfide bridges in multiglobulin multimers. In summary, the results of the analysis (Figure 27-32) are as follows: N-acetyl-L-cysteine and mercaptosuccinic acid distinguish between correct and incorrect disulfide bridges under the conditions used. You can't do it essentially. Glutathione, cysteine ethyl ester and 2-mercaptoethanol were all able to significantly reduce disulfide bridges in the multimeric form within each characteristic range of urea concentration within -10 minutes, at which time the oxidized monomer. β₂-m remains in the oxidized form. Glutathione clearly has the ability to selectively reduce false disulfide bridges at higher urea concentrations compared to cysteine ethyl ester and 2-mercaptoethanol, and therefore among the selected and tested thiols, glutathione. Would be the reducing agent of choice for the periodic regeneration of human β2-microglobulin. As a result of these experiments, the concentration of urea in the reduction buffer B for the regeneration procedure used in Example 13 was reduced from 8M (Example 1) to 6M, and therefore human β.₂-The total regeneration yield of microglobulin was improved from 53% to 87%. Example 13 Regeneration of Purified Human β2-Microglobulin: Comparative Analysis of Three Regeneration Steps A one-pass, stepwise or gradual transition from strong modification and reduction conditions to non-modification and non-reduction conditions. The following series of experiments was undertaken to obtain comparable quantitative data assessing the importance of circulation in terms of regeneration yield for a simple regeneration step with. Purified Recombinant β obtained as described in Example 1.₂-Microglobulin fusion proteins have been reduced and denatured to obtain raw materials that are free of irregularities such as proteolytic products or trace amounts of fusion protein fractions damaged by irreversible oxidation or other chemical induction. First, the optimization step described in Example 12 was used to modify the periodic regeneration conditions described in Example 1 in order to increase the number of production cycles. The optimized regeneration protocol was similar to buffer and other experimental parameters, except that buffer B in this experiment was 6 M urea, 50 mM Tris hydrochloride pH 8, 0.5 M NaCl and 4 mM glutathione. It was the same as that described in Example 1. Using the composition of this buffer B, Ni as described in Example 1.⁺⁺-Loaded NTA-attached to agarose to regenerate 3 batches of pure fusion protein. One batch was subjected to the buffer circulation described in Example 1 and the circulations of batches 2 and 3 were subjected to a simple linear buffer gradient (from 100% B to 0% B over 24 hours) and a step gradient (in one step). It was replaced from 100% B to 0% B, followed by 0% B buffer for 24 hours). In each regeneration experiment, all polypeptide materials were recovered as soluble fractions that could be eluted under non-denaturing conditions and remaining insoluble fractions that could only be eluted under modified and reducing conditions, as described in Example 1. Coomass stains that separate appropriately diluted and measured aliquots of soluble and insoluble fractions under reducing or non-reducing conditions, such as those required to properly separate disulfide-crosslinked monomers from soluble polymers in soluble fractions. Quantitative Concentration Analysis of SDS-PAGE Gels (Hoeffer) The yield of the correctly folded fusion protein was measured using an optical scanner HW from Scientific, CA, USA and a GS-370 concentration analysis SW package). If it was necessary to obtain reliable concentration data for both strong and weak bands in the gel lane, several diluted samples were scanned and analyzed to obtain a reduced data set. Experimental Details and Results Purified denatured and reduced fusion proteins: One batch of human β2-microglobulin fusion proteins was regenerated as described in Example 1. 96% of this fusion protein was recovered in the soluble fraction (Figure 32, Lanes 2-5). 56% of this soluble fraction was in the form of monomeric disulfide bridges. Therefore, the total regeneration efficiency obtained was 53%. A monomeric fusion protein was purified from the multimer by ion exchange chromatography using S-Sepharose (Pharmacia, Sweden). The soluble fraction obtained after regeneration was gel-filtered using Sephadex G-25 (Pharmacia, Sweden) into a buffer containing 5 mM NaCl and 5 mM Tris hydrochloride pH 8, and diluted with water to a 2-fold volume. Then applied to an S-Sepharose column and then eluted with a gradient (5 column volumes from 2.5 mM Tris hydrochloride pH 8 and 2.5 mM NaCl to 25 mM Tris Hydrochloride pH 8 and 100 mM NaCl). The properly folded monomeric fusion protein purified to&gt; 95% purity (Fig. 32, lanes 6 and 7) was then converted to 6M in guanidinium hydrochloride and 0.1M in DTE, 8M urea, 50 mM tris. The gel was filtered into a buffer containing hydrochloric acid pH 8, 1M NaCl and 10 mM 2-mercaptoethanol, and then divided into aliquots for use as raw materials for the regeneration experiments described below. Periodic regeneration of purified fusion protein: 1 aliquot of denatured reduction fusion protein Ni⁺⁺-Loaded NTA columns were then applied and the columns were washed with a buffer containing 1 column volume of 6M guanidinium hydrochloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol. Then buffer A: 50 mM Tris hydrochloride pH 8, 0.5 M NaCl and 3.2 mM / 0.4 mM reduced / oxidized glutathione and buffer B: 50 mM Tris hydrochloride pH 8, 0.5 M NaCl, 6 M urea. And 4 mM reduced glutathione were used to buffer the fusion protein according to the scheme shown in Table 1. After completion of buffer circulation, the fusion protein was recovered in quantitatively soluble form by elution of the column with buffer containing 50 mM Tris hydrochloride pH 8, 0.5 M NaCl and 20 mM EDTA. 87% was obtained in the form of a properly disulfide crosslinked monomer. Regeneration of purified fusion protein by linear gradient: 1 aliquot of denatured reduction fusion protein Ni⁺⁺-Apply to loaded NTA columns, then buffer the column with 1 column volume of 6M guanidinium hydrochloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol, followed by 1 column volume of 50 mM Tris hydrochloride. Washed with buffer containing pH 8, 0.5 M NaCl, 6 M urea and 4 mM reduced glutathione. Then, buffer A: 50 mM Tris hydrochloride pH 8, 0.5 M NaCl and 3.2 mM / 0.4 mM reduced / oxidized glutathione and buffer B: 50 mM Tris hydrochloride pH 8, 0.5 M NaCl, 6 M urea. A 24-hour linear gradient from 100% B to 100% A was applied at 2 ml / min using and 4 mM reduced glutathione. After completion of the gradient, the soluble fraction of the fusion protein was eluted in a buffer containing 50 mM Tris hydrochloride pH 8, 0.5 M NaCl and 20 mM EDTA. The remaining insoluble fraction was extracted from the column in buffer containing 50 mM Tris hydrochloride 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 this soluble fraction was recovered in the form of a properly disulfide crosslinked monomer. Therefore, the total regeneration efficiency obtained was 29% (Fig. 33, lanes 5-7). Regeneration of purified fusion protein by buffer step method: 1 aliquot of denatured reduction fusion protein Ni⁺⁺-Loaded NTA columns were then applied and the columns were washed with a buffer containing 1 column volume of 6M guanidinium hydrochloride, 50 mM Tris hydrochloride pH 8 and 10 mM 2-mercaptoethanol. Then, before recovering the soluble fraction of the fusion protein in a buffer containing 50 mM Tris hydrochloride pH 8, 0.5 M NaCl and 20 mM EDTA, 50 mM Tris Hydrochloride pH 8, 0.5 M NaCl and 3.2 mM / 0.4 A buffer containing mM reduced / oxidized glutathione was applied to the column at 2 ml / min for 24 hours. The remaining insoluble fraction was extracted from the column in buffer containing 50 mM Tris hydrochloride pH 8, 1 M NaCl, 8 M urea, 10 mM 2-mercaptoethanol and 20 mM EDTA. 34% of the fusion protein was recovered in the soluble fraction and 28% of this soluble fraction was recovered in the properly disulfide crosslinked form of the monomer. Therefore, the total regeneration efficiency obtained was 9.5% (Fig. 33, Lanes 1-3). Conclusion In summary, human β as a model protein₂-When using microglobulins, (a) direct buffer optimization prior to periodic regeneration and improved purification of the fusion protein significantly increased regeneration yield (from 53% to 87%). , (B) Continuous denaturation-restoration circulation is otherwise superior to a single regeneration under comparable experimental conditions (87% vs. 29% or 9.5% yield). However, it will be concluded. References<img file="JP3695467B2_D0013.tif" /><img file="JP3695467B2_D0014.tif" />List of sequences (1) General information: (i) Applicant: (A) Name: Denzyme ASP (B) Street: Gastab Wesbay 10 (C) City: Alassie (E) Country: Denmark (F) Postal code : 8000 (ii) Title of Invention: Improved Method for Regenerating Nucleic Acid (iii) Sequence Number: 47 (iv) Computer Readable: (A) Medium Type: Flop Disk (B) Computer: IBM PC Compatible Gender (C) Operating system: PC-DOS / MS-DOS (D) Software: Patent In Release # 1.0, Version # 1.25 (EPO) (2) Information about SEQ ID NO: 1 (i) Sequence characteristics: (A) ) Length: 1554 Base pair (B) Type: Nucleic acid (C) Chain: Double-stranded (D) Topology: Linear (ii) Molecular type: cDNA (iii) Hypothesis: Yes (iii) Antisense: No (vi) ) Origin: (A) Organism: Bos taurus (ix) Features: (A) Name / Key: CDS (B) Position: 76..1551 (xi) Sequence description: SEQ ID NO: 1: 1:<img file="JP3695467B2_D0015.tif" /><img file="JP3695467B2_D0016.tif" /><img file="JP3695467B2_D0017.tif" />(2) Information about SEQ ID NO: 2 (i) Sequence characteristics: (A) Length: 492 Amino acids (B) Type: Amino acids (D) Topology: Linear (ii) Molecular type: Protein (xi) sequence Description: SEQ ID NO: 2:<img file="JP3695467B2_D0018.tif" /><img file="JP3695467B2_D0019.tif" />(2) Information about SEQ ID NO: 3 (i) Sequence characteristics: (A) Length: 42 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 3:<img file="JP3695467B2_D0020.tif" />(2) Information about SEQ ID NO: 4 (i) Sequence characteristics: (A) Length: 22 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 4:<img file="JP3695467B2_D0021.tif" />(2) Information about SEQ ID NO: 5 (i) Sequence features: (A) Length: 44 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 5:<img file="JP3695467B2_D0022.tif" />(2) Information about SEQ ID NO: 6 (i) Sequence features: (A) Length: 23 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 6:<img file="JP3695467B2_D0023.tif" />(2) Information about SEQ ID NO: 7 (i) Sequence features: (A) Length: 40 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 7:<img file="JP3695467B2_D0024.tif" />(2) Information about SEQ ID NO: 8 (i) Sequence features: (A) Length: 26 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 8:<img file="JP3695467B2_D0025.tif" />(2) Information about SEQ ID NO: 9 (i) Sequence features: (A) Length: 39 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 9:<img file="JP3695467B2_D0026.tif" />(2) Information about SEQ ID NO: 10 (i) Sequence characteristics: (A) Length: 26 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 10:<img file="JP3695467B2_D0027.tif" />(2) Information about SEQ ID NO: 11 (i) Sequence features: (A) Length: 42 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 11:<img file="JP3695467B2_D0028.tif" />(2) Information about SEQ ID NO: 12 (i) Sequence features: (A) Length: 30 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 12:<img file="JP3695467B2_D0029.tif" />(2) Information about SEQ ID NO: 13 (i) Sequence features: (A) Length: 29 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 13:<img file="JP3695467B2_D0030.tif" />(2) Information about SEQ ID NO: 14 (i) Sequence features: (A) Length: 32 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 14:<img file="JP3695467B2_D0031.tif" />(2) Information about SEQ ID NO: 15 (i) Sequence features: (A) Length: 42 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 15:<img file="JP3695467B2_D0032.tif" />(2) Information about SEQ ID NO: 16 (i) Sequence features: (A) Length: 29 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 16:<img file="JP3695467B2_D0033.tif" />(2) Information about SEQ ID NO: 17 (i) Sequence features: (A) Length: 41 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 17:<img file="JP3695467B2_D0034.tif" />(2) Information about SEQ ID NO: 18 (i) Sequence features: (A) Length: 29 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 18:<img file="JP3695467B2_D0035.tif" />(2) Information about SEQ ID NO: 19 (i) Sequence features: (A) Length: 46 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 19:<img file="JP3695467B2_D0036.tif" />(2) Information about SEQ ID NO: 20 (i) Sequence features: (A) Length: 29 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 20:<img file="JP3695467B2_D0037.tif" />(2) Information about SEQ ID NO: 21 (i) Sequence features: (A) Length: 44 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 21:<img file="JP3695467B2_D0038.tif" />(2) Information about SEQ ID NO: 22 (i) Sequence features: (A) Length: 30 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 22:<img file="JP3695467B2_D0039.tif" />(2) Information about SEQ ID NO: 23 (i) Sequence features: (A) Length: 42 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 23:<img file="JP3695467B2_D0040.tif" />(2) Information about SEQ ID NO: 24 (i) Sequence features: (A) Length: 30 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 24:<img file="JP3695467B2_D0041.tif" />(2) Information about SEQ ID NO: 25 (i) Sequence features: (A) Length: 47 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 25:<img file="JP3695467B2_D0042.tif" />(2) Information about SEQ ID NO: 26 (i) Sequence features: (A) Length: 30 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 26:<img file="JP3695467B2_D0043.tif" />(2) Information about SEQ ID NO: 27 (i) Sequence features: (A) Length: 46 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 27:<img file="JP3695467B2_D0044.tif" />(2) Information about SEQ ID NO: 28 (i) Sequence features: (A) Length: 33 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 28:<img file="JP3695467B2_D0045.tif" />(2) Information about SEQ ID NO: 29 (i) Sequence features: (A) Length: 41 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 29:<img file="JP3695467B2_D0046.tif" />(2) Information about SEQ ID NO: 30 (i) Sequence features: (A) Length: 31 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 30:<img file="JP3695467B2_D0047.tif" />(2) Information about SEQ ID NO: 31 (i) Sequence features: (A) Length: 40 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 31:<img file="JP3695467B2_D0048.tif" />(2) Information about SEQ ID NO: 32 (i) Sequence features: (A) Length: 26 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 32:<img file="JP3695467B2_D0049.tif" />(2) Information about SEQ ID NO: 33 (i) Sequence features: (A) Length: 39 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 33:<img file="JP3695467B2_D0050.tif" />(2) Information about SEQ ID NO: 34 (i) Sequence features: (A) Length: 25 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 34:<img file="JP3695467B2_D0051.tif" />(2) Information about SEQ ID NO: 35 (i) Sequence features: (A) Length: 38 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 35:<img file="JP3695467B2_D0052.tif" />(2) Information about SEQ ID NO: 36 (i) Sequence features: (A) Length: 29 base pairs (B) Type: Nucleic acid (C) Chain: Single chain (D) Topology: Linear (ii) Molecular type: DNA (synthesis) (xi) Sequence description: SEQ ID NO: 36:<img file="JP3695467B2_D0053.tif" />(2) Information about SEQ ID NO: 37 (i) Sequence features: (A) Length: 6 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 37:<img file="JP3695467B2_D0054.tif" />(2) Information about SEQ ID NO: 38 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 38:<img file="JP3695467B2_D0055.tif" />(2) Information about SEQ ID NO: 39 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 39:<img file="JP3695467B2_D0056.tif" />(2) Information about SEQ ID NO: 40 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 40:<img file="JP3695467B2_D0057.tif" />(2) Information about SEQ ID NO: 41 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 41:<img file="JP3695467B2_D0058.tif" />(2) Information about SEQ ID NO: 42 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 42:<img file="JP3695467B2_D0059.tif" />(2) Information about SEQ ID NO: 43 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 43:<img file="JP3695467B2_D0060.tif" />(2) Information about SEQ ID NO: 44 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 44:<img file="JP3695467B2_D0061.tif" />(2) Information about SEQ ID NO: 45 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 45:<img file="JP3695467B2_D0062.tif" />(2) Information about SEQ ID NO: 46 (i) Sequence features: (A) Length: 4 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 46:<img file="JP3695467B2_D0063.tif" />(2) Information about SEQ ID NO: 47 (i) Sequence features: (A) Length: 6 amino acids (B) Type: Amino acid (C) Chain: Single chain (D) Topology: Linear (ii) molecule Type: Peptide (xi) Sequence Description: SEQ ID NO: 47:<img file="JP3695467B2_D0064.tif" />(2) Information about SEQ ID NO: 48 (i) Sequence features: (A) Length: 15 Amino acids (B) Type: Amino acids (C) Chains: Single-stranded (D) Topology: Linear (ii) Molecules Type: Peptide (xi) Sequence Description: SEQ ID NO: 48:<img file="JP3695467B2_D0065.tif" />(2) Information about SEQ ID NO: 49 (i) Sequence features: (A) Length: 119 Amino acids (B) Type: Amino acids (C) Chains: Single-stranded (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 49:<img file="JP3695467B2_D0066.tif" />(2) Information about SEQ ID NO: 50 (i) Sequence Features: (A) Length: 119 Amino Acids (B) Type: Amino Acids (C) Chains: Single Chains (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 50:<img file="JP3695467B2_D0067.tif" />(2) Information about SEQ ID NO: 51 (i) Sequence Features: (A) Length: 217 Amino Acids (B) Type: Amino Acids (C) Chains: Single Chains (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 51:<img file="JP3695467B2_D0068.tif" />(2) Information about SEQ ID NO: 52 (i) Sequence Features: (A) Length: 4544 Amino Acids (B) Type: Amino Acids (C) Chains: Single Chains (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 52:<img file="JP3695467B2_D0069.tif" /><img file="JP3695467B2_D0070.tif" /><img file="JP3695467B2_D0071.tif" /><img file="JP3695467B2_D0072.tif" /><img file="JP3695467B2_D0073.tif" /><img file="JP3695467B2_D0074.tif" /><img file="JP3695467B2_D0075.tif" /><img file="JP3695467B2_D0076.tif" /><img file="JP3695467B2_D0077.tif" /><img file="JP3695467B2_D0078.tif" /><img file="JP3695467B2_D0079.tif" /><img file="JP3695467B2_D0080.tif" /><img file="JP3695467B2_D0081.tif" /><img file="JP3695467B2_D0082.tif" /><img file="JP3695467B2_D0083.tif" /><img file="JP3695467B2_D0084.tif" />(2) Information about SEQ ID NO: 53 (i) Sequence features: (A) Length: 487 Amino acids (B) Type: Amino acids (C) Chains: Single-stranded (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 53:<img file="JP3695467B2_D0085.tif" /><img file="JP3695467B2_D0086.tif" /><img file="JP3695467B2_D0087.tif" />(2) Information about SEQ ID NO: 54 (i) Sequence Features: (A) Length: 790 Amino Acids (B) Type: Amino Acids (C) Chains: Single Chains (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 54:<img file="JP3695467B2_D0088.tif" /><img file="JP3695467B2_D0089.tif" /><img file="JP3695467B2_D0090.tif" />(2) Information about SEQ ID NO: 55 (i) Sequence Features: (A) Length: 153 Amino Acids (B) Type: Amino Acids (C) Chains: Single Chains (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 55:<img file="JP3695467B2_D0091.tif" />(2) Information about SEQ ID NO: 56 (i) Sequence Features: (A) Length: 202 Amino Acids (B) Type: Amino Acids (C) Chains: Single Chains (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 56:<img file="JP3695467B2_D0092.tif" />(2) Information about SEQ ID NO: 57 (i) Sequence Features: (A) Length: 246 Amino Acids (B) Type: Amino Acids (C) Chains: Single Chains (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 57:<img file="JP3695467B2_D0093.tif" />(2) Information about SEQ ID NO: 58 (i) Sequence Features: (A) Length: 101 Amino Acids (B) Type: Amino Acids (C) Chains: Single Chains (D) Topology: Linear (ii) Molecules Type: Protein (xi) Sequence description: SEQ ID NO: 58:<img file="JP3695467B2_D0094.tif" />