Fusion proteins

Description

[0001] The present invention relates to non-cytotoxic fusion proteins and their therapeutic use as analgesic molecules.

[0002] Toxins can generally be divided into two groups depending on the type of effect they have on the target cell. More specifically, the first group of toxins kill their natural target cells: they are therefore referred to as cytotoxic toxin molecules. Examples of this group of toxins include, but are not limited to, plant toxins such as ricin and abrin, and bacterial toxins such as diphtheria toxin and Pseudomonas exotoxin A. Cytotoxic toxins are of great interest in the development of "magic missiles (e.g., immunoconjugates, comprising a cytotoxic toxin component and an antibody that binds to a specific marker on a target cell) for the treatment of cellular disorders and diseases such as cancer. Cytotoxic toxins typically kill target cells by inhibiting cellular protein synthesis processes.

[0003] The second group of toxins, known as non-cytotoxic toxins, do not kill (according to their name) their natural target cells. Non-cytotoxic toxins have been dealt with much less in the commercial context than their cytotoxic counterparts: they exert their effect on a target cell by inhibiting cellular processes other than protein synthesis. Non-cytotoxic toxins are produced by many plants and many microorganisms such as Clostridium sp. and Neisseria sp.

[0004] Clostridial neurotoxins are proteins whose molecular weight is typically in the order of 150 kDa. They are produced by various species of bacteria, especially of the genus Clostridium, most notably C. tetani, and several strains of C. botulinum, C. butyricum and C. argentinense. At present, eight different classes of Clostridial neurotoxins are known, namely: tetanus toxin and botulinum neurotoxin of serotypes A, B, Cl, D, E, F and G. They all share a similar structure and mode of action.

[0005] Clostridial neurotoxins are a major group of non-cytotoxic toxin molecules. They are produced by host bacteria as single polypeptides which undergo post-translational modification by proteolytic cleavage to form two polypeptide chains linked by a disulfide bond. These two chains are referred to as the heavy chain (H chain), having a molecular weight of about 100 kDa, and the light chain (L chain), having a molecular weight of about 50 kDa.

[0006] The L chains show a protease function (zinc-dependent endopeptidase activity) and a high substrate specificity for vesicle and / or plasma membrane associated proteins involved in exocytosis. L-chains of different species or serotypes of Clostridium can hydrolyze different but specific peptide bonds in one of three substrate proteins, namely synaptobrevin, syntaxin or SNAP-25. These substrates are important components of the secretion processes in the neurosecretory system.

[0007] Neisseria sp., Primarily of the species N. gonorrhoeae, produces functionally similar non-cytotoxic proteases. An example of such a protease is the IgA protease (see WO99 / ​​58571).

[0008] It has been well documented in the art that toxin molecules can be redirected to a cell that is not the toxin's natural target cell. In such redirection, the modified toxin is capable of binding to the desired target cell and, upon subsequent translocation into the cytosol, is capable of exerting its effect on the target cell. Such redirection is achieved by replacing the natural targeting moiety (TM) with another TM. In this regard, the TM is chosen such that it binds to the desired target cell and allows the modified toxin to pass later to the endosome in the target cell. The modified toxin also contains a translocation domain that allows the entry of a non-cytotoxic protease into the cytosol of the cell. The translocation domain may be a natural toxin translocation domain or another translocation domain derived from a microbial protein with translocation activity.

[0009] WO94 / 21300 describes, for example, modified Clostridial neurotoxin molecules capable of regulating the density of an Integral Membrane Protein (IMP) present on the surface of a target cell. Thus, the modified neurotoxin molecules are capable of regulating the activity (e.g., glucose uptake) of the target cell. WO96 / 33273 and WO99 / ​​17806 describe modified clostridial neurotoxin molecules that target peripheral sensory afferents. The modified neurotoxin molecules are therefore capable of exerting an analgesic effect. WO00 / 10598 describes the production of modified Clostridial neurotoxin molecules to target cells that exhibit mucus excess (or neurons that control those cells that exhibit excess mucus); these modified neurotoxins are capable of inhibiting excessive secretion by these cells. WO01 / 21213 describes modified Clostridial neurotoxin molecules that target many different types of target cells other than nerve cells. The modified molecules are thus able to prevent secretion from target cells. Additional publications in the field of redirecting toxin molecules include: WO00 / 62814, WO00 / 04926, US5,773,586, WO93 / 15766, WO00 / 61192, and WO99 / ​​58571.

[0010] The above-mentioned TM replacement can be achieved by conventional chemical coupling techniques well known to the skilled person. This is mentioned in Hermanson, GT (1996), Bioconjugate technigues, Academic Press, and Wong, SS (1991), Chemistry of protein conjugation and cross-linking, CRC Press.

[0011] Chemical conjugation is, however, often imprecise. Following conjugation, for example, the TMs may link to the remainder of the conjugate at more than one site.

Chemical conjugation is also difficult to control. For example, the TM may link to the remainder of the modified toxin at the linkage site on the protease component and / or on the translocation component. This is a problem when only one of these ingredients (preferably at a single site) needs to be combined to achieve therapeutic efficacy.

[0013] Chemical conjugation thus results in a mixed population of modified toxin molecules, which is undesirable.

[0014] Instead of chemical conjugation, TM replacement may be performed by recombinantly producing a single polypeptide fusion protein (see WO98 / 07864). The technique relies on an in vivo bacterial mechanism to produce the native Clostridial neurotoxin (i.e., holotoxin) to produce a fusion protein with the following structure:

NH2- [protease component] - [translocation component] - [TM] -COOH

[0015] According to WO98 / 07864, the TM is placed towards the C-terminus of the fusion protein. The fusion protein is then activated by treatment with a protease that cleaves in place between the protease component and the translocation component. Thus, a two-chain protein is formed, having the protease component as a single polypeptide chain, covalently attached (via a disulfide bridge) to a second single polypeptide chain containing the plus TM translocation component. While the method described in

WO98 / 07864 corresponds (with respect to the structural system of the fusion protein) to the natural Clostridial holotoxin expression system, the present inventors found that this system may result in the production of certain fusion proteins with significantly reduced binding capacity to the intended target cell.

[0016] There is therefore a need for an alternative or improved system for constructing a non-cytotoxic fusion protein.

[0017] The present invention addresses one or more of the above-mentioned problems by providing a single chain polypeptide fusion protein as defined in the claims appended hereto.

[0018] US 5,989,585 describes Clostridial redirected neurotoxin therapeutics which bind to peripheral sensory afferents. These therapeutic agents are useful as analgesics.

[0019] WO2004 / 024909 describes the preparation of recombinant toxin fragments lacking a functional H-binding domain._c Clostridium's natural neurotoxin. These molecules therefore lack the native ability to bind to the natural Clostridial neurotoxin. WO 2004/024909 also describes recombinant toxin fragments which may include a non-Clostridium TM located at the C terminus of the H translocation domain._N.

[0020] EP1422240 describes peptide nociceptin analogs.

[0021] WO2005 / 023309 describes a counter-intuitive approach to producing therapeutics based on Clostridial redirected neurotoxin. More specifically, while the described therapeutic agents act by inhibiting secretion processes in cells, the counter-intuitive aspect relies on the selection of a TM, which in fact stimulates cell secretion from the target cell of interest.

[0022] The system of WO98 / 07864 works well to generate conjugates having a TM that requires a C-terminal domain to interact with a binding site on the target cell. In this regard, WO98 / 07864 describes fusion proteins with a C-terminal domain that is "free, that is, available for interaction with a binding site on a target cell. The present inventors have found that this structural arrangement is not suitable for all TMs. One such category of TM is the group of TMs that bind to nociceptive sensory afferents. More specifically, the present inventors found that the system of the WO 98/07864 fusion protein is not optimal for TMs requiring an N-terminal domain to interact with a binding site on a nociceptive sensory afferent. This problem is especially serious with TMs that require a specific N-terminal amino acid residue or a specific amino acid residue sequence, including an N-terminal amino acid residue, to interact with a binding site on a nociceptive sensory afferent.

[0023] In contrast to WO98 / 07864, the present invention provides a system for the production of non-cytotoxic conjugates in which the TM component of the conjugate has a suitable internal domain binding domain or an amino acid sequence located in the center (i.e. relative to the linear peptide sequence) of the TM, or preferably, located towards the N-terminus of the TM, or more preferably at or near the N-terminus. The N-terminal domain is capable of binding to a binding site on a nociceptive sensory afferent, and the TM preferably requires that a specific and defined sequence of amino acid residue or residues be free at the N-terminus.

[0024] The non-cytotoxic protease component of the present invention is a non-cytotoxic protease or a fragment thereof, said protease or protease fragment capable of cleaving different but specific peptide bonds in one of three protein substrates, namely synaptobrevin, syntaxin or SNAP -25, the exocytic fusion apparatus in a nociceptive sensory afferent. These substrates are important components of the neurosecretory system. The non-cytotoxic protease component of the present invention is preferably a Neisseria IgA protease or a fragment thereof or a Clostridial neurotoxin L chain or fragment thereof. A particularly preferred non-cytotoxic protease component is the botulinum neurotoxin (BoNT) L chain or fragment thereof.

[0025] The translocation component of the present invention enables the translocation of a non-cytotoxic protease (or a fragment thereof) into a target cell such that the cytosol of the target cell is functionally expressing the protease activity. The translocation component is preferably capable of forming ion-permeable pores in lipid membranes under low pH conditions. Preferably, it has been found that only those portions of the protein molecule should be used which are capable of forming pores within the endosomal membrane. The translocation component may be obtained from a microbial protein source, in particular a bacterial or viral protein source. Thus, in one embodiment, the translocation component is a translocating domain of an enzyme such as a bacterial toxin or viral protein. The translocation component of the present invention is preferably a Clostridial neurotoxin H chain or fragment thereof. Most preferably it is an H domain_N (or a functional component thereof), with H._N is a portion or fragment of the H chain of a Clostridial neurotoxin corresponding roughly to the amino terminal half of the H chain or a domain corresponding to this fragment in an intact H chain.

[0026] The TM component of the present invention is responsible for the binding of the conjugate of the present invention to a binding site on the target cell. The TM component is thus simply the ligand through which the conjugate of the present invention binds to a selected target cell.

[0027] In the context of the present invention, the target cell is a nociceptive sensory afferent, preferably a primary nociceptive afferent (for example A fiber such as δ fiber or C fiber). The conjugates of the present invention are thus capable of inhibiting the release of a neurotransmitter or neuromodulator [e.g., glutamate, substance P, calcitonin gene related peptide (CGRP) and / or neuropeptide Y] from distinct populations of nociceptive sensory afferents. The use of conjugates reduces or eliminates the transmission of sensory afferent signals (e.g., neurotransmitters or neuromodulators) from peripheral to central pain conducting fibers, so they have utility as therapeutic molecules in the treatment of pain, especially chronic pain.

[0028] It is routinely confirmed that the TM binds to a nociceptive sensory afferent. For example, a simple radioactive displacement experiment may be performed in which tissue or cells representative of a nociceptive sensory afferent (e.g. DRG) are exposed to labeled (e.g. tritium) ligand in the presence of an excess of unlabeled ligand. In such an experiment, the relative ratio of nonspecific and specific binding can be assessed, thereby confirming that the ligand binds to a target cell of a nociceptive sensory afferent. Optionally, the test may involve using one or more binding antagonists and observing the decrease in ligand binding capacity. Examples of this type of experience can be found in Hulme,

EC (1990), Receptor-binding studies, a brief outline, pp. 303-311, in Receptor biochemistry, A Practical Approach, ed. EC Hulme, Oxford University Press.

[0029] The fusion proteins of the present invention generally show a reduced binding affinity (up to nearly 100-fold) for target cells of the nociceptive sensory afferent as compared to the corresponding "free TMs." Nevertheless, the fusion proteins of the present invention surprisingly show good efficiency. This can be attributed to two main factors. First, the non-cytotoxic protease component has catalytic properties, whereby the therapeutic effect of several of these molecules is rapidly enhanced. Second, the receptors present on the nociceptive sensory afferents need only act as a gateway to regulate entry of the therapeutic, and need not be stimulated to the level necessary to achieve a pharmacological response mediated by ligand-receptor interaction. Accordingly, the fusion proteins of the present invention may be administered at doses much lower than would be required for other types of painkillers such as NSAIDs (non-steroidal anti-inflammatory drugs), morphine and gabapentin. The latter molecules are typically administered in amounts corresponding to many micrograms or milligrams (even up to hundreds of milligrams), while the fusion proteins of the present invention can be administered at significantly lower doses, typically at least 10 fold lower, more typically 100 fold lower.

[0030] The TM preferably has a maximum of 50 amino acid residues, more preferably a maximum of 40 amino acid residues, particularly preferably a maximum of 30 amino acid residues and most preferably a maximum of 20 amino acid residues.

[0031] Opioids are a preferred group of TMs of the present invention. This family of peptides includes enkephalins (met and leu), endomorphins 1 and 2, β-endorphin, and dynorphin. Opioid peptides are often used in medicine to modify activity against nociceptors and other cells involved in the pain response. Opioids can be included in the pharmacotherapy of chronic cancer and non-cancer pain on all three steps of the World Health Organization pain ladder, highlighting their importance in pain management. When the term "opioids" is referred to, it includes fragments, variants and derivatives thereof which retain the ability to bind to nociceptive sensory afferents. [0032] The TM of the present invention may also be a molecule that acts as an "agonist at one or more receptors present on a nociceptive sensory afferent, especially on a primary nociceptive afferent." Conventionally, an agonist is any molecule that can increase or decrease an activity within a cell, namely any molecule that simply causes a change in cell activity. The conventional meaning of the word "agonist" would include, for example, a chemical capable of binding to a receptor on a cell and inhibiting a reaction or activity, or a drug inducing an active response by activating the receptors, whether the response is to increase or decrease cellular activity.

[0033] For the purposes of this invention, however, an agonist is more specifically defined as a molecule capable of stimulating an exocytic fusion process in a target cell; this process can be inhibited by a protease (or fragment thereof) capable of cleaving the protein of the exocytic fusion apparatus in that target cell.

[0034] Accordingly, the agonist definition of the present invention as set forth herein would exclude many molecules that would be conventionally considered agonists. For example, nerve growth factor (NGF) is an agonist - with respect to its ability to facilitate neuronal differentiation by binding to the TrkA receptor. However, NGF is not an agonist when assessed according to the above criteria as it is not a major inducer of exocytic fusion. In addition, the NGF-stimulated process (ie, cell differentiation) is not susceptible to inhibition by the protease activity of the non-cytotoxic toxin molecule.

[0035] The agonist properties of a TM that binds to a receptor on a nociceptive afferent can be confirmed by the methods described in Example 10.

[0036] In a preferred embodiment of the invention, the target for the TM is the ORL1 receptor. This receptor belongs to the class of G protein-coupled receptors and has a structure with seven transmembrane domains. The properties of the ORL1 receptor are discussed in detail in Mogił and Pasternak (2001), Pharmacological Reviews, Vol. 53, No. 3, pp. 381415.

In one embodiment, the TM is a molecule that binds (preferably, specifically binds) to an ORL1 receptor. More preferably, the TM is an "ORL1 receptor agonist." The term "agonist in this context is defined above.

The agonist properties of a TM which binds to ORL1 receptor can be confirmed by the methods described in Example 10. The methods are based on previous experiments [see Inoue et al. 1998 [Proc. Natl. Acad. Sci., 95, 10949-10953]), which confirm that the natural ORL1 receptor agonist nociceptin induces substance P release from nociceptive primary afferents. The argument is that:

> - nociceptin-induced responses are reversed by specific NK1 receptor (substance P receptor) antagonists;

> - pre-treatment of cells with capsaicin (which reduces the amount of substance P in small diameter primary afferents) reduces the responses induced by nociceptin.

[0039] Similarly, Inoue et al. Confirm that injection of botulinum neurotoxin type A into the plantar abrogates the nociceptin-induced responses. Since BoNT is known to inhibit the release of substance P from primary afferents (Welch et al., 2000, Toxicon, 38, 245-258), this supports the relationship between nociceptin and ORL, the interaction and the subsequent release of substance P.

[0040] It can therefore be said that the TM exhibits agonist activity at the ORL receptor_lz if TM induces the release of substance P from nociceptive sensory afferents (see Example 10).

[0041] In a particularly preferred embodiment of the invention, the TM is nociceptin - a natural ligand for the ORL1 receptor. Nociceptin targets the ORL1 receptor with high affinity. Examples of other preferred TMs include:

<td>Code</td><td>Sequence</td><td>Item writings.</td><td>Seq ID No.</td>

<td>Nociceptin 1-17</td><td>FGGFTGARKSARKLANQ</td><td> [1]</td><td> 37,38</td>

<td>Nociceptin 1-11</td><td>FGGFTGARKSA</td><td> [1]</td><td> 39,40</td>

<td>Nociceptin [Y10] 1111</td><td>FGGFTGARKYA</td><td> [1]</td><td> 41,42</td>

<td>N ocy ceptin [Y11] 1 - 11</td><td>FGGFTGARKSY</td><td> [1]</td><td> 43,44</td>

<td>Nociceptin [Y14] I17</td><td>FGGFTGARKSARKYANQ</td><td> [1]</td><td> 45,46</td>

<td>Nociceptin 1-13</td><td>FGGFTGARKSARK</td><td> [2]</td><td> 47,48</td>

<td>Nociceptin [R14K15] 1-17 (determined also in this description as "variant" nociceptins)</td><td>FGGFTGARKSARKRKNQ</td><td> [3,4]</td><td> 49,50</td>

<td>Peptide agonist</td><td>Peptide agonists from the combinatorial library method</td><td> [5]</td><td> -</td>

[1] Mogił and Pasternak, 2001, Pharmacol. Rev. 53, 381-415

[2] Maile et al., 2003, Neurosci. Lett., 350, 190-192

[3] Rizzi et al., 2002, J. Pharmacol. Exp. Therap., 300, 57-63

[4] Okada et al., 2000, Biochem. Biophys. Res. Commun., 278, 493-498

[5] Dooley et al., 1997, J Pharmacol Exp Ther. 283 (2), 735-41.

[0042] The above identified "TM variant has a particularly good binding affinity (compared to natural nociceptin) for nociceptive sensory afferents. This is unexpected since the modification of the amino acids occurs remote from the N-terminus of the TM. In addition, the modifications are located almost at the C-terminus of the TM which in turn is linked to a large polypeptide sequence (i.e., the translocation domain). Overall, a TM-containing fusion protein will show an approximately 100-fold reduction in binding capacity relative to TM alone. The aforementioned "TM variant by itself exhibits an approximate 3-10 fold increase in binding capacity to a nociceptive sensory afferent (e.g. via the ORL1 receptor) compared to native nociceptin. Thus, it can be expected that a fusion containing a "TM variant will show an approximately 10-fold reduction in binding capacity to a nociceptive sensory afferent (via for example an ORL1 receptor) compared to" free nociceptin. " The present inventors have, however, found that such fusion proteins, containing a "TM variant, have a binding capacity that surprisingly closely resembles the binding capacity of" free nociceptin " Fig. 14.

In the context of the present invention, the term "opioid or" ORL1 receptor agonist (such as nociceptin or any of the peptides listed in the table above) includes molecules exhibiting at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology with a given opioid or agonist. The agonist homologues retain the agonist properties of nociceptin at the ORL1 receptor, which can be tested using the methods described in Example 10. Likewise, an opioid homologue essentially retains the opioid binding function to which it exhibits high homology.

[0044] The invention also includes fragments, variants and derivatives of any of the TMs described above. These fragments, variants and derivatives essentially retain the properties attributed to these TMs.

[0045] In addition to the opioid and non-opioid classes of TM mentioned above, many other polypeptides are suitable for targeting the conjugates of the present invention to nociceptive sensory afferents (e.g., nociceptors). In this regard, galanin and galanin derivatives should be mentioned in particular. Galanin receptors are pre- and postsynaptically present in DRGs (Liu and Hokfelt, (2002), Trends Pharm. Sci., 23 (10), 468-74) and their expression is increased in states of neuropathic pain. Proteinase activated receptors (PAR), especially PAR-2, are also a preferred group of TMs of the present invention. PAR-2 ​​agonists are known to induce / cause acute inflammation, in part through a neurogenic mechanism. PAR2 is expressed in primary spinal afferents and PAR2 agonists stimulate the release of substance P (SP) and calcitonin gene-related peptide (CGRP) in peripheral tissues.

[0046] A particularly preferred collection of TMs according to the present invention comprises the following substances:

<td>Ligand</td><td>References</td>

<td>Nociceptin</td><td>Guerrini et al. (1997) J. Med. Chem., 40, pp. 1789-1793</td>

<td>β-endorphma</td><td>Blanc et al. (1983) J. Biol. Chem., 258 (13), pp. 82778284</td>

<td>Endomorphin 1; Endomorphin 2</td><td>Zadina, et al., (1997). Nature, 386, pp. 499-502</td>

<td>Dynorfma</td><td>Fields and Basbaum (2002) Chapter 11, in: The Textbook of pain, Wall &amp; Melzack (eds.).</td>

<td>Met-enkephalin</td><td>Fields and Basbaum (2002) Chapter 11, in: The Textbook of pain, Wall &amp; Melzack (eds.).</td>

<td>Leu-enkephalin</td><td>Fields and Basbaum (2002) Chapter 11, in The Textbook of pain, Wall &amp; Melzack (eds.).</td>

<td>Galanina</td><td>Xu et al. (2000) Neuropeptides, 34 (3i4), 137-147</td>

<td>PAR-2 ​​peptide</td><td>Vergnolle et al. (2001) Nat. Med., 7 (7), 821-826</td>

[0047] The protease cleavage site of the present invention allows cleavage (preferably controlled cleavage) of the fusion protein at a position between the non-cytotoxic protease component and the TM component. It is in this cleavage reaction that the fusion protein is converted from a single chain polypeptide to a disulfide-linked double chain polypeptide.

[0048] According to a preferred embodiment of the present invention, the TM is bound via a domain or amino acid sequence located far from the C-terminus of the TM. A suitable binding domain may include, for example, an internal domain or an inwardly located amino acid sequence (i.e., with respect to a linear peptide sequence) of a TM. Preferably, a suitable binding domain is located towards the N-terminus of the TM, more preferably at or near the N-terminus of the N-terminus.

[0049] In one embodiment, the single chain fusion polypeptide may contain more than one proteolytic cleavage site. However, if there are two or more such sites, they are different, which essentially prevents multiple cleavage events from occurring in the presence of a single protease. In another embodiment, it is preferred that the single chain fusion polypeptide comprises a single protease cleavage site.

[0050] The protease cleavage sequence (s) may be introduced (and / or deleted) at the DNA level by conventional means, for example by site-directed mutagenesis. Screening for the presence of cleavage sequences can be performed manually or using computer software (e.g., the MapDraw program from DNASTAR, Inc.).

[0051] While any protease cleavage site may be used, the following are preferred:

Enterokinase (DDDDKj.)

Factor Xa (IEGR f / IDGR f)

TEV (tobacco etch virus (ENLYFQj, G))

Thrombin (LVPRj, GS)

PreScission (LEVLFQ J.GP).

[0052] The term "protease cleavage site also includes an intein, which is a self-cleavage sequence. The self-assembly reaction can be controlled by using, for example, a variable concentration of the reducing agent.

[0053] In this application, the protease cleavage site is cleaved whereby the N-terminal region (preferably the N-terminus) of the TM is exposed. The resulting polypeptide comprises a TM with an N-terminal or internal domain substantially free of conjugate residues. This arrangement ensures that the N-terminal component (or internal domain) of the TM can directly interact with the binding site on the target cell.

[0054] The TM and protease cleavage site are separated in the fusion protein by at most zero amino acid residues. After cleavage at the protease cleavage site, a TM conjugate is obtained, containing an N-terminal domain substantially free of conjugate residues. This arrangement ensures that the N-terminal TM component can directly interact with a binding site on the target cell.

[0055] One advantage associated with the above-mentioned activation step is that the TM only becomes susceptible to N-terminal degradation after proteolytic cleavage of the fusion protein. In addition, the selection of a specific protease cleavage site allows for the selective activation of a polypeptide fusion into a two-chain conformation.

[0056] When producing the single chain fusion polypeptide of the present invention, the protease cleavage site is positioned between the TM and the non-cytotoxic protease component.

[0057] Preferably, in the single chain fusion, the TM is between the protease cleavage site and the translocation component. This ensures that the TM is attached to the translocation domain (i.e., as in native Clostridial holotoxin), although in the case of the present invention the order of the two components is reversed relative to the native holotoxin. A further advantage of this arrangement is that the TM is located in the exposed loop region of the fusion protein, exerting minimal structural effects on the conformation of the fusion protein. In this respect, this loop is referred to by various terms such as: linker, activation loop, inter-domain linker, or simply a surface exposed loop (Schiavo et al. 2000, Phys. Rev., 80, 717-766; Turton et al. , 2002, Trends Biochem. Sci., 27, 552-558).

[0058] In one embodiment, in the single polypeptide chain, the non-cytotoxic protease component and the translocation component are linked by a disulfide bond. After cleavage at the protease cleavage site, the polypeptide therefore adopts a two-chain conformation in which the protease and translocation components remain linked together by a disulfide bond. For this purpose, it is preferred that the protease and translocation components remain spaced apart in the single chain fusion protein by a maximum of 100 amino acid residues, more preferably a maximum of 80 amino acid residues, particularly preferably a maximum of 60 amino acid residues, and most preferably a maximum of 50 amino acid residues.

[0059] In one embodiment, the non-cytotoxic protease component forms a disulfide bond with the translocation component of the fusion protein. The amino acid residue of the protease component which forms the disulfide bond is, for example, within the last 20, preferably within the last 10, C-terminal amino acid residues of the protease component. Similarly, the amino acid residue within the translocation component that forms the second portion of the disulfide bond may be within the first 20, preferably the first 10 N-terminal amino acid residues of the translocation component.

[0060] Alternatively, in a single chain polypeptide, the non-cytotoxic protease component and the TM may be linked together by a disulfide bond. In this regard, the amino acid residue of the TM, forming the disulfide bond, is preferably beyond the N-terminus of the TM, more preferably towards the C-terminus of the TM.

[0061] In one embodiment, the non-cytotoxic protease component forms a disulfide bond with the TM component of the fusion protein. In this regard, the amino acid residue of the protease component, forming the disulfide bond, is preferably within the last 20, more preferably within the last 10 C-terminal amino acid residues of the protease component. Similarly, the amino acid residue within the TM component forming the second part of the disulfide bond is preferably within the last 20, more preferably within the last 10 C-terminal amino acid residues of the TM.

[0062] The above disulfide bonding system has the advantage that the protease and translocation components are arranged similarly to the native clostridial neurotoxin. For comparison, with respect to the primary amino acid sequence of native Clostridial neurotoxin, the corresponding cysteine ​​amino acid residues are 8 to 27 amino acid residues apart, according to Popoff, MR, and Marvaud, JC, 1999, Structural and genome features of clostridial neurotoxins, Chapter 9, in: The Comprehensive Sourcebook of Bacterial Protein Toxins, edited by Alouf and Freer:

<td>Serotype¹</td><td colspan="2">Sequence</td><td>"Native" length between CC</td>

<td>BoNT / Al</td><td colspan="2">CVRGIITSKTKS— LDKGYNKALNDLC</td><td> 23</td>

<td>BoNT / A2</td><td colspan="2">CVRGIIPFKTKS— LDEGYNKALNDLC</td><td> 23</td>

<td>BoNT / B</td><td>CKSVKAPG -----------</td><td>--------- ic</td><td> 8</td>

<td>BoNT / C</td><td>CHKAIDGRS ----------</td><td>-LYNKTLDC</td><td> 15</td>

<td>BoNT / D</td><td>CLRLTK ----------------</td><td>-NSRDDSTC</td><td> 12</td>

<td>BoNT / E</td><td>CKN-IVSVK -----------</td><td>GIRK — SIC</td><td> 13</td>

<td>BoNT / F</td><td>CKS-VIPRK -----------</td><td>GTKAPP-RLC</td><td> 15</td>

<td>BoNT / G</td><td>CKPYMYKNT --------</td><td>—GKSE — QC</td><td> 13</td>

<td>Awning</td><td colspan="2">CKKIIPPTNIRENLYNRTASLTDLGGE LC</td><td> 27</td>

<td>information</td><td colspan="2">only from proteolytic strains</td><td></td>

[0063] The fusion protein may contain one or more purification tags located N-terminal to the protease component and / or C-terminal to the translocation component.

[0064] Any purification tag may be used, but the following are preferred:

His tag (e.g. 6 x histidine), preferably as a C-terminal tag and / or as an N-terminal MBP tag (maltose binding protein), preferably as an N-terminal tag, GST tag (glutathione-S-transferase), preferably as an N-tag His-MBP tag, preferably as N-terminal tag, GST-MBP tag, preferably as N-terminal tag, thioredoxin tag, preferably as N-terminal tag, CBD tag (chitin binding domain), preferably as N-terminal tag.

[0065] According to a further embodiment of the present invention, one or more peptide spacer molecules may be included in the fusion protein. For example, a peptide spacer may be placed between the purification tag and the remainder of the fusion protein molecule (e.g., between the N-terminal purification tag and the protease component of the present invention and / or between the C-terminal purification tag and the translocation component of the present invention). A peptide spacer may also be inserted between the TM and the translocation components of the present invention.

[0066] A variety of spacer molecules may be used in any of the fusion proteins of the present invention. Examples of such spacer molecules include those illustrated in Figures 28 and 29. Particular mention should be made of GS15, GS20, GS25 and Hx27 - see. Figures 28 and 29.

[0067] The present inventors have surprisingly found that the fusion proteins (e.g. CPNv / A) of the present invention can exhibit improved binding activity to nociceptive sensory afferents when the spacer size is selected such that (when used) the C TM end and The N of the translocation component are spaced apart by 40-105 angstroms, preferably 50-100 angstroms, and more preferably 50-90 angstroms. In another embodiment, the preferred spacers have an amino acid sequence of 11-29 amino acid residues, preferably 15-27 amino acid residues, and more preferably 2027 amino acid residues. Preferred spacers can be routinely identified and obtained according to Crasto, CJ and Feng, JA (2000) May, 13 (5), pp. 309-312 - see. also http: //www.fccc/edu/research/ labs / feng / Iimker.html.

[0068] According to a second aspect of the present invention, there is provided a DNA sequence encoding the above-mentioned single chain polypeptide. In a preferred aspect of the present invention, the DNA sequence is produced as part of a DNA vector, wherein the vector comprises a promoter and a terminator.

[0069] In a preferred embodiment, the vector comprises a promoter selected from the following:

Promoter Agent for Typical conditions of induction induction

Tac (hybrid) IPTG 0.2 mM (0.05-2.0 mM)

AraBAD L-arabinose 0.2% (0.002-0.4%)

T7 operator - IPTG 0.2 mM (0.05-2.0 mM) lac

[0070] The DNA construct of the present invention is preferably developed in silico and then synthesized by conventional DNA synthesis techniques.

[0071] The aforementioned DNA sequence information is optionally modified for codon biasing depending on the final expression pattern in the host (e.g. E. coli) cells. [0072] The DNA backbone is preferably screened for any natural nucleic acid sequences which, when transcribed and translated, would yield amino acid sequences corresponding to a protease cleavage site encoded by the sequence encoding the second peptide. Such screening can be performed manually or using computer software (e.g., the MapDraw program from DNASTAR, Inc.).

[0073] According to a further embodiment of the present invention, there is provided a method of producing a non-cytotoxic agent, comprising:

a. contacting a fusion protein of the present invention that is a single chain polypeptide with a protease capable of cleavage at the protease cleavage site;

b. cleavage at the protease cleavage site, thereby forming a two-chain fusion protein.

[0074] According to this aspect, there is provided a two-chain polypeptide that generally mimics the structure of a Clostridial holotoxin. More specifically, the resultant two-chain polypeptide typically has a structure in which:

a. the first chain comprises a non-cytotoxic protease or a fragment thereof, said protease or protease fragment being capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent;

b. the second chain comprises a TM and a translocation domain capable of translocating a protease or protease fragment from within the endosome, across the endosomal membrane, into the cytosol of a nociceptive sensory afferent; and the first and second chains are linked to each other by a disulfide bond.

[0075] According to a further aspect of the present invention, there is provided the use of a single chain or double chain polypeptide of the invention in the manufacture of a medicament for the treatment, prevention or alleviation of pain.

[0076] The present invention may be used in the management of a wide spectrum of pain-related conditions and diseases, particularly chronic pain related diseases. The preferred diseases include cancer pain and non-cancer pain, inflammatory pain and neuropathic pain. The opioid fusions of the present invention are particularly suitable for use in inflammatory pain, but may be less applicable for neuropathic pain. Galanin fusions are better suited for use in neuropathic pain.

[0077] In this application, the polypeptides of the present invention are typically used in the form of a pharmaceutical composition in association with a pharmaceutical carrier, diluent and / or excipient, although the particular form of the composition may be adapted to the mode of administration. The agent is preferably administered to a mammal, more preferably a human.

[0078] The polypeptides may, for example, be used in the form of a sterile solution for intra-articular or intracranial administration. Spinal injection (e.g., into the epidural space or into the fluid spaces) is preferred.

[0079] The dose range for administration of the polypeptides of the present invention is that range which will achieve the desired therapeutic effect. It should be noted that the range of necessary dosages depends on the particular type of ingredients, route of administration, type of preparation, age of the patient, type, extent or severity of the patient's disease, contraindications (if any) and at the discretion of the attending physician.

[0080] A suitable daily dose range is 0.00011 mg / kg, preferably 0.0001-0.5 mg / kg, more preferably 0.002-0.5 mg / kg, particularly preferably 0.004-0.5 mg / kg. The unit dose may be from less than 1 microgram to 30 mg, but will typically be in the range of 0.01 to 1 mg; such a dose may be administered daily or preferably less frequently, for example every week or every six months.

[0081] A particularly preferred dosing regimen is based on the administration of 2.5 ng of the fusion protein (e.g. CPNv / A) as dose IX. In this regard, preferred doses will be in the range 1Χ-100Χ (ie 2.5-250 ng). This dose level is significantly less (i.e. at least 10 times, typically 100 times less) than would be the level that would be used with other types of analgesic molecule such as NSAIDs, morphine and gabapentin. Moreover, the aforementioned difference greatly increases when the same comparison is made on a mole basis - and this is because the molecular weight of the fusion proteins of the present invention is much greater than that of conventional "small therapeutic molecules".

[0082] Significant variation in dosage levels, however, can be expected depending upon the particular ingredients used and the differences in the effectiveness of the individual routes of administration.

[0083] Dosage level variability can be corrected using standard empirical optimization methods as known in the art.

[0084] Injectable compositions can be prepared as solutions, suspensions, or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle before use.

[0085] Liquid dosage unit forms are typically prepared utilizing a pyrogen-free, sterile vehicle. The active ingredients, depending on the vehicle and concentration used, can be dissolved or suspended in the vehicle.

[0086] In preparing dosing solutions, the polypeptides may be dissolved in a vehicle, the solution made isotonic, if necessary with the addition of sodium chloride, and sterile filtered through a sterile filter using aseptic techniques. The solution is then poured into suitable sterile vials or ampoules and sealed. Alternatively, if the stability of the solution is adequate, the solution can be sterilized in sealed containers by autoclaving them.

[0087] Advantageously, auxiliary agents such as buffering, solubilizing, stabilizing, preserving or bactericidal, suspending or emulsifying agents can be dissolved in the carrier.

[0088] Dry powders, which are dissolved or suspended in a suitable vehicle before use, can be prepared by filling the pre-sterilized drug substance and other ingredients in a sterile container using aseptic technique in a sterile environment.

[0089] Alternatively, the polypeptides and other ingredients may be dissolved in an aqueous vehicle, the solution sterilized by filtration, and distributed into suitable containers using aseptic technique in a sterile environment. The product is then lyophilized and the containers sealed under aseptic conditions.

[0090] Parenteral suspensions are similarly prepared, suitable for intramuscular, subcutaneous, or intradermal injection, except that the sterile components are suspended in a sterile vehicle instead of dissolving them, and sterilization cannot be accomplished by filtration. The components may be isolated aseptically or may alternatively be sterilized after isolation by, for example, gamma irradiation.

[0091] Preferably, the composition includes a suspending agent, for example polyvinylpyrrolidone, to aid uniform distribution of the ingredients.

Definition of e

[0092] A Targeting Moiety (TM) is any chemical structure associated with an agent that interacts functionally with a binding site, resulting in a physical relationship between the agent and the surface of a target cell. In the context of the present invention, the target cell is a nociceptive sensory afferent. The term "TM includes any molecule (i.e., a naturally occurring molecule or a chemically / physically modified variant thereof) that is able to bind to a binding site on a target cell, where that binding site is internalizable (e.g., to form an endosome) - which is also called receptor dependent endocytosis. The TM may exhibit an endosomal membrane translocation function: in which case it is not necessary to have separate components - the TM and the translocation domain - present in the agent of the present invention.

[0093] The TM of the present invention binds (preferably binds specifically) to a nociceptive sensory afferent (e.g., a primary nociceptive afferent). In this regard, "bind specifically means that the TM binds to a nociceptive sensory afferent (e.g., a primary nociceptive afferent) with greater affinity than to other neurons, such as non-nociceptive afferents, and / or to motor neurons (i.e. with the natural target of the holotoxin - the clostridium neurotoxin). The term "specific binding can also mean that the TM binds to a given receptor, for example an ORL1 receptor, with a binding affinity (K_and) of 10^d M.^-1 or more, preferably 10⁷ M.^-1 or more, more preferably 10⁸ M.^-1 or more, most preferably 10⁹ M "¹ or more .

[0094] For the purposes of this invention, an agonist is defined as a molecule capable of stimulating an exocytic fusion process in a target cell, which process is susceptible to inhibition by a protease (or fragment thereof) capable of cleaving the protein of the exocytic fusion apparatus in the target cell.

[0095] Accordingly, the definition of a particular agonist according to the present invention will exclude many molecules which would be conventionally considered to be agonists.

[0096] Nerve growth factor (NGF) is for example an agonist with respect to its ability to facilitate neuronal differentiation by binding to the TrkA receptor. However, NGF is not an agonist when assessed according to the criteria set out above, as it is not a major inducer of exocytic fusion. In addition, the NGF-stimulated process (i.e., cell differentiation) is not inhibited by the protease activity of the non-cytotoxic toxin molecule.

[0097] The term "fragment, when used in relation to a protein, means a peptide containing at least thirty-five, preferably at least twenty-five, more preferably at least twenty, and most preferably at least ten amino acid residues of the protein in question.

[0098] The term "variant, when used in reference to a protein, means a peptide or peptide fragment of a protein containing one or more amino acid analogs (e.g., a non-natural amino acid) or a substitution conjugate.

[0099] The term "derivative", when used in reference to a protein, means a protein that is composed of the protein in question and an additional peptide sequence. Preferably, this additional peptide sequence does not interfere with the basic folding, ie does not adversely affect the conformational structure of the starting protein. Two or more peptides (or fragments or variants) can be linked together to form a derivative. Alternatively, the peptide (or fragment or variant) can be linked to an unrelated molecule (e.g., a second, unrelated peptide). The derivatives can be made by chemical synthesis, but are typically prepared by recombinant nucleic acid methods. Additional ingredients such as lipids and / or polysaccharides and / or polyketides may be included.

[0100] Throughout this specification, the term "ORL1 receptor" includes all receptors of the ORL1 family. The ORLi family of receptors typically have the structure of seven transmembrane domains and are coupled to G proteins of the G ± and Go families. Example 12 describes the determination of the G protein stimulating activity of ORL1 receptor ligands. Example 11 describes how to measure the reduction in cell cAMP levels following ORLi activation. Another feature of the ORL1 family of receptors is that they are typically capable of binding nociceptin (the natural ORLi ligand). All alternative splicing variants of the ORL1 receptor belong, for example, to the ORL1 receptor family.

[0101] The term "non-cytotoxic" means that the protease molecule in question does not kill the target cell to which it is redirected.

[0102] The term proteases according to the present invention includes all naturally occurring non-cytotoxic proteases capable of cleaving one or more proteins of the exocytic fusion apparatus in eukaryotic cells.

[0103] The protease of the present invention is preferably a bacterial protease (or a fragment thereof). More preferably, the bacterial protease is selected from the genus Clostridium or Neisseria (for example Clostridium L chain or Neisseria IgA protease, preferably from N. gonorrhoeae).

[0104] The present invention also includes modified non-cytotoxic proteases containing non-naturally occurring amino acid sequences and / or synthetic amino acid residues, provided that the modified proteases still exhibit the above-mentioned protease activity.

[0105] The protease of the present invention preferably exhibits serine or metalloprotease activity (e.g. endopeptidase activity). The protease is preferably specific for a SNARE protein (such as for example SNAP-25, synaptobrevin / VAMP or syntaxin).

[0106] Particular attention should be paid to the protease domains of neurotoxins, for example the protease domains of bacterial neurotoxins. The present invention thus encompasses the use of naturally occurring neurotoxin domains as well as recombinantly produced versions of these naturally occurring neurotoxins.

[0107] Exemplary neurotoxins are produced by bacilli of the genus Clostridium; the term "Clostridial neurotoxin" includes the neurotoxins produced by C. tetani (TeNT) and the AG serotypes of C. botulinum (BoNT), as well as the closely related BoNTneurotoxins produced by C. baratii and C. butyricum. The abbreviations listed above are used throughout this specification. The term "BoNT / A means, for example, that the source of the neurotoxin is BoNT (serotype A). Similar rules apply to the nomenclature of the other BoNT serotypes.

[0108] The term "L chain fragment" means a component of the L chain of a neurotoxin, the fragment having metalloprotease activity and is capable of proteolytic cleavage of a vesicle and / or plasma membrane associated protein involved in cellular exocytosis.

[0109] A translocation domain is a molecule that enables a protease (or a fragment thereof) to be translocated into a target cell so that functional expression of the protease activity occurs within the cytosol of the target cell. Whether any molecule (e.g. protein or peptide) exhibits the necessary translocation function of the present invention can be confirmed by any of a number of conventional tests.

[0110] Shone C. (1987) describes, for example, an in vitro assay using liposomes that is exposed to a test molecule. The presence of the necessary translocation function is confirmed by the release from K liposomes⁺ and / or labeled with NAD, which can be easily monitored [see Shone C. (1987) Eur. J. Biochem; bar 167 (1): pp. 175-180].

[0111] Another example is provided by Blaustein R. (1987): this publication describes a simple in vitro assay using planar, bilayer phospholipid membranes. The membranes are exposed to the test molecule and the necessary translocation function is confirmed by the increase in conductivity through these membranes [see Blaustein (1987) FEBS LettS; bar 226, no. 1: pp. 115-120].

[0112] Additional methods for evaluating membrane fusion and thereby identifying translocation domains suitable for use in the present invention are described in Methods in Enzymology Vol. 220 and 221, Membrane Fusion Technigues, Parts A and B, Academic Press 1993.

[0113] The translocation domain is preferably capable of forming ion-permeable pores in lipid membranes under low pH conditions. Preferably, it has been found that only those portions of the protein molecules that are capable of forming pores within the endosomal membrane can be used.

[0114] The translocation domain may be obtained from microbial protein sources, especially bacterial or viral protein sources. Thus, in one embodiment, a translocation domain is a translocating domain of an enzyme such as a bacterial toxin or viral protein.

[0115] As is well known from the literature, certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of fusion proteins expressed in membrane viruses are capable of forming such pores. Such domains can be used according to the present invention.

[0116] The translocation domain may be derived from a Clostridium, namely it may be an H domain_N (or its functional component). H._N is a portion or fragment of the H chain of a Clostridial neurotoxin approximately corresponding to the amino terminal half of the H chain, or a domain corresponding to this fragment in an intact H chain. Preferably, the H chain is substantially devoid of the natural H component binding function_c For the H chain, the function H_ccan be eliminated by deleting the H amino acid sequence_c (at the DNA synthesis level or at the post-synthesis level by treatment with nuclease or protease). Instead, the function H._c can be deactivated by chemical or biological treatment. The H chain is therefore preferably incapable of binding to a binding site on a target cell to which native clostridial neurotoxin (i.e., holotoxin) binds.

[0117] In one embodiment, the translocation domain is an H domain_N (or a fragment thereof) of a Clostridial neurotoxin. Examples of suitable clostridial translocation domains include:

Botulinum neurotoxin type A - amino acid residues (449-871)

Botulinum neurotoxin type B - amino acid residues (441-858)

Botulinum neurotoxin type C - amino acid residues (442-866)

Type D botulinum neurotoxin - amino acid residues (446-862)

Type E botulinum neurotoxin - amino acid residues (423-845)

Botulinum neurotoxin type F - amino acid residues (440-864)

Botulinum neurotoxin type G - amino acid residues (442-863)

Tetanus neurotoxin - amino acid residues (458-879)

[0118] Further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani can be found in Henderson et al. (1997), The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0119] The term "H_N includes naturally occurring portions of the neurotoxin H._N and modified parts of H._N with amino acid sequences that do not occur in nature and / or contain synthetic amino acid residues, provided that the modified H portions_Nstill exhibit the translocation function mentioned above.

[0120] Alternatively, the translocation domain may be derived from a source other than Clostridium (see Table 4). Examples of a translocation domain from sources other than Clostridia include, but are not limited to, a diphtheria toxin translocation domain [O'Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269,22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], a translocation domain from the Pseudomonas type A exotoxin [Prior et al. Biochemistry (1992) 31, 3555-3559], translocation domains from anthrax toxin [Blanka et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], a variety of fusogenic or hydrophobic peptides having a translocating function [Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp. 7934-7938] and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp. 1986-1992]. A translocation domain may mirror a translocation domain present in a naturally occurring protein or may include amino acid variations as long as this does not eliminate the ability of the translocation domain to cause translocation.

[0121] Specific examples of viral translocation domains suitable for use in the present invention include certain translocating domains derived from viral-expressed membrane fusion proteins. In the publications of Wagner et al. (1992) and Murata et al. (1992), for example, describes the translocation function (i.e., causing membrane fusion and vesicle formation) of many fusogenic and amphiphilic peptides derived from the N-terminal region of influenza virus hemagglutinin - To other membrane virus-expressed fusion proteins known to exhibit the desired translocating activity, include the translocating domain of the Semliki Forest virus (SFV) fusogenic peptide, vesicular stomatitis virus (VSV) G glycoprotein translocating domain, SER virus F protein translocating domain and foam virus envelope glycoprotein translocating domain. Virus-encoded Aspike proteins, for example the E1 protein of SFV and the VSV G protein, find particular use in the context of the present invention.

[0122] Use of the Translocation Domains listed in the Table (below) includes the use of sequence variants thereof. A variant may contain one or more conservative nucleic acid substitutions and / or nucleic acid deletions or insertions, provided that the variant exhibits the necessary translocating function. A variant may also contain one or more amino acid substitutions and / or amino acid deletions or insertions, provided that the variant has the necessary translocating function.

<td>Translocation domain source</td><td>Amino acid residues</td><td>Literature</td>

<td>Diphtheria toxin</td><td> 194-380</td><td>Silverman et al., 1994, J. Biol. Chem. 269, 22524-22532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51</td>

<td>Pseudomonas exotoxin domain II</td><td> 405-613</td><td>Prior et al., 1992, Biochemistry 31, 35553559 Kihara and Pastan, 1994, Bioconj Chem. 5, 532-538</td>

<td>Influenza virus haemagglutinin</td><td>GLFGAIAGFIENGWE GMIDGWYG and its variants</td><td>Plank et al., 1994, J. Biol. Chem. 269, 1291812924 Wagner et al., 1992, PNAS, 89, 79347938 Murata et al., 1992, Biochemistry 31, 1986-1992</td>

<td>Semliki Forest virus fusogenic protein</td><td>Translocation domain</td><td>Kielian et al., 1996, J Cell Biol. 134 (4), 863-872</td>

<td>G-glycoprotein of vesicular stomatitis virus</td><td> 118-139</td><td>Yao et al., 2003, Virology 310 (2), 319332</td>

<td>The F protein of the SER virus</td><td>Translocation domain</td><td>Seth et al., 2003, J Virol 77 (11) 6520-6527</td>

<td>The envelope glycoprotein of the foam virus</td><td>Translocation domain</td><td>Picard-Maureau et al., 2003, J Virol. 77 (8), 4722-4730</td>

Figures

[0123]

Fig. 1 Purification of the LC / A-nociceptinH fusion protein_n/AND

Figure 2 Purification of the reference molecule of the nociceptin-LC / AH fusion protein_N/AND

Figure 3 Purification of the LC / C-nociceptinH fusion protein_n/ C

Fig. 4 Purification of LC / A-met enkephalin-H fusion protein_N/AND

Fig. 5 Comparison of the binding efficiency of the LC / A-nociceptin-H fusion protein_N/ A and nociceptin-LC / AH fusion protein_N/AND

Fig. 6 In vitro catalytic activity of LC / A-nociceptin-H fusion protein_N/AND

Fig. 7 Purification of the LC / A-nociceptin-H variant fusion protein_N/AND

Fig. 8 Comparison of the binding efficiency of the LC / A-nociceptin-H fusion protein_N/ A and LC / A-variant nociceptin-H fusion protein_N/AND

Fig. 9 Expressed / purified product (s) from the LC / A-nociceptin-H fusion protein family_N/ A with a variable length cutout

Figure 10 Inhibition of SP release and SNAP-25 cleavage by CPN-A

Fig. 11 Inhibition of SP release and SNAP-25 cleavage over a prolonged time after DRG exposure to CPN-A

Fig. 12 Clearing of SNAP-25 by CPNv-A

Fig. 13 Cleavage of SNAP-25 for a prolonged time after DRG exposure to CPNv-A

Fig. 14 CPNv-A fusion dependent displacement of the binding [³H] -nociceptin

Fig. 15 Expressed / purified CPNv (Ek) -A product

Fig. 16 Clearing SNAP-25 by CPNv (Ek) -A

Fig. 17 Expressed / purified CPNv-C product

Fig. 18 Cleavage of syntaxin by CPNv-C

Fig. 19 CPN-A efficacy in a model of acute capsaicin-induced mechanical allodynia

Fig. 20 Efficacy of CPN-A in the streptozotocin (STZ) model of diabetic neuropathy (neuropathic pain)

Figure 21 Efficacy of CPNv-A in a model of acute capsaicin-induced mechanical allodynia

Fig. 22 Expressed / purified LC / A-CPLE-H product_n/AND

Fig. 23 Expressed / purified LC / A-CPBE-H product_n/AND

Fig. 24 Expressed / purified CPOP-A product

Fig. 25 Expressed / purified CPOPv-A product

Figure 26 Cleavage of SNAP-25 in vitro in a DRG cell model

Fig. 27 Expressed / purified CPNv-A-FXa-HT (removable his tag)

Figure 28 In vitro efficacy of LC / Anocyceptin-H fusion protein_N/ A with variable spacer length, as assessed by a ligand competition assay

Fig. 29 In vitro efficacy of LC / Anocyceptin-H fusion proteins_N/ A with variable spacer length as assessed by in vitro SNAP-25 cleavage.

[0124] Below the figures will be described in more detail.

Figure 1 - Purification of the LC / A nocyc ep tyin-H fusion protein_N/AND

[0125] Using the methodology set out in Example 9, the LC / A-nociceptin-H fusion protein was purified from E. coli BL21 cells._N/AND. Briefly, the soluble products obtained after breaking the cell structure were applied to a nickel charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with factor Xa to activate the fusion protein and remove the maltose binding protein (MBP) tag, then reapplied onto a second nickel charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (panel A) and Western (panel B). Anti-nociceptin immune sera (obtained from Abcam) were used as the primary antibody for Western analysis. The final, purified material in the absence and presence of a reducing agent is identified in the lanes labeled [-] and [+], respectively.

Figure 2 - Purification of the reference molecule nociceptin-LC / AH fusion protein_N/AND

[0126] Using the methodology set out in Example 9, the nociceptin-LC / A-HN / A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained after breaking the cell structure were applied to a nickel charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with factor Xa to activate the fusion protein and remove the maltose binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (panel A) and Western (panel B). Anti-nociceptin immune sera were used as the primary antibody for Western analysis. The final, purified material in the absence and presence of a reducing agent is identified in the paths marked [-] and [+], respectively

Figure 3 - Purification of LC / Cnocyc ep tyin-H fusion protein_N/ C

[0127] Using the methodology set out in Example 9, the LC / C-nociceptin-HN / C fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained after breaking the cell structure were applied to a nickel charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with factor Xa to activate the fusion protein and remove the maltose binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (panel A) and Western (panel B). Anti-nociceptin immune sera (obtained from Abcam) were used as the primary antibody for Western analysis. The final, purified material in the absence and presence of a reducing agent is identified in the lanes labeled [-] and [+], respectively.

Figure 4 - Purification of LC / A-met enkephalin-H fusion protein_N/AND

[0128] Using the methodology set forth in Example 9, the LC / A-met enkephalin-H fusion protein was purified from E. coli BL21 cells._N/AND. Briefly, the soluble products obtained after breaking the cell structure were applied to a nickel charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with factor Xa to activate the fusion protein and remove the maltose binding protein (MBP) tag, then reapplied onto a second nickel charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE. The final, purified material in the absence and presence of a reducing agent is identified in the paths marked [-] and [+], respectively

Figure 5 - Comparison of the binding efficiency of the LC / A-nociceptin-H fusion protein_N/ A and nocyceptin-LC / AH fusion protein_N/AND

[0129] The ability of the nociceptin fusion to bind to the ORLi receptor was assessed by a simple competition assay. Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of test material in the presence of 1 nM [³H] -nociceptin. The reduction in the specific binding of the radiolabeled ligand was assessed using a scintillation counter and a graph was drawn to compare the effectiveness of the unlabeled ligand (Tocris nociceptin). It is clear that the LC / A-nociceptin-H_N/ A is far more advantageous than the nociceptin-LC / AH fusion_N/ A in interaction with the ORLi receptor.

Figure 6 - In vitro catalytic activity of LC / A-nociceptin-H fusion protein_N/AND

[0130] The in vitro endopeptidase activity of the purified LC / A-nociceptin-H fusion protein was assessed._N/ A, essentially as described in Chaddock et al. 2002, Prot. Express Purif. 25, 219-228. Briefly, SNAP-25 peptide immobilized on an ELISA plate was exposed to varying concentrations of the fusion protein for 1 hour at 37 ° C. After the washing series, the cleaved SNAP-25 peptide was quantified based on its reactivity with specific antisera.

Figure 7 - Purification of the LC / A-variant nocycepin-H fusion protein_N/AND

[0131] Using the methodology set out in Example 9, the LC / A-Nociceptin-H variant fusion protein was purified from E. coli BL21 cells._N/AND. Briefly, the soluble products obtained after breaking the cell structure were applied to a nickel charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with factor Xa to activate the fusion protein and remove the maltose binding protein (MBP) tag, then reapplied onto a second nickel charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE. The final, purified material in the absence and presence of a reducing agent is identified in the paths marked [-] and [+], respectively

Figure 8 - Comparison of the binding efficiency of the LC / A-nociceptin-H fusion protein_N/ A and LC / A-variant nociceptin-H fusion protein_N/AND

[0132] The ability of the nociceptin fusion to bind to the ORLi receptor was assessed by a simple competition assay. Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of test material in the presence of 1 nM [³H] -nociceptin. The reduction in the specific binding of the radiolabeled ligand was assessed using a scintillation counter and a graph was drawn comparing the effectiveness of the unlabeled ligand (Tocris nociceptin). It is clear that the LC / A-nociceptin-H variant fusion_N/ A (CPNvLHA) is more favored than LC / A fusion-nociceptin-H variant_N/ A (CPN-LHA) by interaction with the ORLi receptor.

Figure 9 - Expressed / purified product (s) of the LC / A-nociceptin-H fusion protein family_N/ A with a variable length cutout

[0133] Using the methodology set out in Example 9, LC / A-CPN-H fusion variants were purified from E. coli cell paste._n/ A, consisting of GS10, GS30 and ΗΧ27. Samples from LC / A-CPN (GS10) -H purification_n/ A, LC / ACPN (GS15) -H_n/ A, LC / A-CPN (GS25) -H_n/ A, LC / A-CPN (GS30) -H_n/ A and LC / A-CPN (ΗΧ27) -H_N/ A was assessed by SDS-PAGE and then stained with Coomassie Blue. The electrophoretic profile shows the purification of the two-chain substance with disulfide bonds with the expected molecular weight of CPBE-A. Top panel: M = reference molecular weight markers; S = total E. coli protein soluble fraction; FT = proteins not binding to charged Ni²⁺ a sepharose column; FUSION = fusion protein eluted by the addition of imidazole. Bottom panel: Lane 1 = standard molecular weight markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = Clean material after initial capture on charged Ni²⁺a sepharose column; Lane 4 = Factor Xa treated material prior to final capture on charged Ni²⁺ a sepharose column; Lane 5 = purified final material post activation with Factor Xa (5 μΐ); Lane 6 = purified final material post activation with Factor Xa (10 µΐ); Lane 7 = purified final material post activation with Factor Xa (20 μΐ); Lane 8 = purified final material post activation with Factor Xa + DTT (5 µΐ); Lane 9 = purified final material post activation with Factor Xa + DTT (10 µΐ); Lane 10 = purified final material post activation with Factor Xa + DTT (20 µΐ).

Figure 10 - Inhibition of SP release and SNAP-25 cleavage by CPN-A

[0134] Briefly, primary dorsal root ganglion (DRG) cultures were exposed to varying concentrations of CPN-A for 24 hours. Cellular proteins were separated by SDS-PAGE and analyzed by Western blotting using an anti-SNAP-25 probe to facilitate evaluation of SNAP25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis and plotted against the fusion concentration (dashed line). Material was also recovered for the analysis of substance P content using a specific EIA kit. The inhibition of substance P release is illustrated by the solid line. The fusion concentration needed to achieve 50% of maximal SNAP-25 cleavage is estimated to be 6.30 ± 2.48 nM.

Fig. 11 - Inhibition of SP release and SNAP-25 cleavage over a prolonged time after DRG exposure to CPN-A

[0135] Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of CPN-A for 24 hours. Botulinum neurotoxin (BoNT / A) was used as a control. After this pre-exposure, extracellular material was removed by washing and cells were incubated at 37 ° C for various times. Cellular proteins were separated at specific time points by SDS-PAGE and analyzed by Western blotting using an anti-SNAP-25 probe to aid in the evaluation of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis as illustrated by the dotted lines. Material was also recovered for the analysis of substance P content using a specific EIA kit. The inhibition of substance P release is illustrated by the solid lines.

Fig. 12 - Clearing SNAP-25 by CPNv-A

[0136] Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of CPNv-A for 24 hours. Cellular proteins were separated by SDS-PAGE and analyzed by Western blotting using an anti-SNAP-25 probe to facilitate evaluation of SNAP25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. The fusion concentration required to achieve 50% of maximal SNAP25 cleavage is estimated to be 1.38 0.36 nM.

Figure 13 - Cleavage of SNAP-25 for a prolonged time after exposure to DRG to CPNv-A

[0137] Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of CPNv-A for 24 hours. CPN-A was used as a control. After this pre-exposure, extracellular material was removed by washing and cells were incubated at 37 ° C for various times. Cellular proteins were separated by SDS-PAGE at predetermined time points and analyzed by Western blotting with an anti-SNAP-25 probe to aid in assessing SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis.

Figure 14 - CPNv-A fusion dependent displacement of the binding [³H] -nociceptin

[0138] The ability of the nociceptin fusion to bind to the ORLi receptor was assessed by a simple competition assay. Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of test material in the presence of 1 nM [³H] -nociceptin. The reduction in the specific binding of the radiolabeled ligand was assessed using a scintillation counter and a graph was drawn to compare the effectiveness of the unlabeled ligand (Tocris nociceptin). It is clear that the LC / A-nociceptin-H variant fusion_N/ A (denoted as CPNv-LHnA) is more preferred than the LC / A-nociceptin-H fusion_N/ A (labeled CPN-LHnA) for interaction with the ORLi receptor.

Figure 15 - Expressed / purified CPNv (Ek) -A product

[0139] Proteins were subjected to SDS-PAGE then stained with Coomassie blue. The electrophoretic profile shows the purification of the two-chain substance with disulfide bonds with the expected molecular weight of CPNv (Ek) -A. Lane 1 = standard molecular weight markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = Clean material after initial capture on charged Ni²⁺a sepharose column; Lane 4 = purified final material post activation with enterokinase (5 µΐ); Lane 5 = purified final material post activation with enterokinase (10 µΐ); Lane 6 = purified final material post activation with enterokinase (20 µΐ); Lane 7 = purified final material post activation with enterokinase + DTT (5 µΐ); Lane 8 = purified final material post activation with enterokinase + DTT (10 µΐ); Lane 9 = purified final material after activation with enterokinase + DTT (20 μΐ)

Fig. 16 - Clearing SNAP-25 by CPNv (Ek) -A

[0140] Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of CPNv (Ek) -A for 24 hours. Cellular proteins were separated by SDS-PAGE and analyzed by Western blotting using an anti-SNAP-25 probe to facilitate evaluation of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. For comparison, CPNv-A prepared according to Example 9 was used. The percentage of SNAP-25 cleavage by CPNv (Ek) -A (denoted as activated En) and CPNv-A (denoted as activated Xa) is illustrated.

Figure 17 - Expressed / purified CPNv-C product

[0141] Proteins were subjected to SDS-PAGE then stained with Coomassie blue. The electrophoretic profile shows the purification of a two-chain substance with disulfide bonds with the expected molecular weight of CPNvC. Lane 1 = standard molecular weight markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = Clean material after initial capture on charged Ni²⁺ a sepharose column; Lane 4 = Factor Xa treated material prior to final capture on charged Ni²⁺ a sepharose column; Lane 5 = purified material after second capture on Ni-charged Sepharose; Lane 6 = final, cleaned material; Lane 7 = final purified material + DTT; Lane 8 = standard molecular weight markers.

Figure 18 - Cleavage of syntaxin by CPNv-C

[0142] Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of CPNv-C for 24 hours. Cellular proteins were separated by SDS-PAGE, analyzed by Western blotting using an anti-syntaxin probe to facilitate evaluation of syntaxin cleavage. The percentage of cleaved syntaxin was calculated by densitometric analysis. The fusion concentration required to achieve 50% maximal syntaxin cleavage is estimated to be 3.13 ± 1.96 nM.

Figure 19 - CPN-A efficacy in an acute capsaicin-induced mechanical allodynia model. LC / A-nociceptin-H fusion capacity was assessed._N/ A (CPN / A) for inhibition of capsaicin-induced mechanical allodynia following subcutaneous injection into the plantar of the rat hind paw. The paw withdrawal frequency in test animals (PWF%) was assessed in response to a 10 g series of von Frey filament stimuli (10 stimuli x 3 trials) prior to study entry (pre-treatment), after subcutaneous injection of CPN / A to the footbed, but prior to dosing capsaicin (before CAP) and after capsaicin exposure after CPN / A injection (mean response after 15 'and 30'; CAP). Capsaicin exposure was achieved by injecting 10 μΐ 0.3% solution. Sample dilutions were made in 0.5% BSA / saline.

Figure 20 - Efficacy of CPN-A in the Streptozotocin (STZ) model of diabetic neuropathy (neuropathic pain)

[0144] Male Sprague-Dawley rats (250-300 g) were administered 65 mg / kg STZ in citrate buffer (iv) and blood glucose and lipids were determined weekly to assess model readiness. The Paw Withdrawal Threshold (PWT) in response to a series of stimuli with von Frey filaments over a specified period of time is determined. Allodynia is considered to have developed when the PWT of two consecutive measurements, one week apart, is less than 6 g on the scale. At this point, the rats are randomized to the saline group (negative efficacy control), the gabapentin group (positive efficacy control) or the test group (CPN / A). Test materials (20-25 µΐ) are injected subcutaneously as a single injection (except gabapentin) and PWT is measured on day 1 after treatment and periodically thereafter over a 2 week period. Gabapentin (30 mg / kg ip, volume of each injection: 3 ml / kg) is injected daily, 2 hours before the start of the PWT tests.

Fig. 21 - CPNv-A efficacy in a model of acute capsaicin-induced mechanical allodynia. LC / A-variant nociceptin-H fusion capacity was assessed._N/ A (CPNv / A) for inhibition of capsaicin-induced mechanical allodynia following subcutaneous injection into the plantar of the rat hind paw. The paw withdrawal frequency (PWF%) was assessed in test animals in response to a 10 g series of von Frey filament stimuli (10 stimuli x 3 trials) prior to enrollment (pre-treatment), after subcutaneous injection of CPNv / A to the plantar but prior to dosing capsaicin (before CAP) and after capsaicin exposure after CPNv / A injection (mean response after 15 'and 30'; CAP). Capsaicin exposure was achieved by injecting 10 μΐ 0.3% solution. Sample dilutions were made in 0.5% BSA / saline. These data are expressed as the normalized difference in the rate of paw withdrawal where the difference between the peak response (after capsaicin) and the baseline response is

5Ί (before capsaicin) is expressed as a percentage. In this analysis, it can be seen that CPNv / A has a stronger effect than CPN / A, since a lower dose of CPNv / A is sufficient to obtain a similar analgesic effect to that of CPN / A.

Fig. 22 - Expressed / purified LC / A-CPLE-H product_n/AND

[0146] Proteins were subjected to SDS-PAGE and then stained with Coomassie blue. The electrophoretic profile shows the purification of the two-chain substance with disulfide bonds of the expected CPLEA molecular weight. Lane 1 = standard molecular weight markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = Clean material after initial capture on charged Ni²⁺ a sepharose column; Lane 4 = Factor Xa treated material prior to final capture on charged Ni²⁺ a sepharose column; Lane 5 = Clean material after second capture on charged Ni²⁺ a sepharose column; Lane 6 = final, cleaned material; Lane 7 = final purified material + DTT.

Fig. 23 - Expressed / purified LC / A-CPBE-H product_n/AND

[0147] Proteins were subjected to SDS-PAGE and then stained with Coomassie blue. The electrophoretic profile shows the purification of the two-chain substance with disulfide bonds of the expected molecular weight of CPBEA. Lane 1 = total E. coli protein soluble fraction; Lane 2 = Clean material after initial capture on charged Ni²⁺ a sepharose column; Lane 3 = Factor Xa treated material prior to final capture on charged Ni²⁺ a sepharose column; Lane 4 = purified final material post activation with Factor Xa (5 μΐ); Lane 5 = purified final material post activation with Factor Xa (10 μΐ); Lane 6 = purified final material post activation with Factor Xa (20 μΐ); Lane 7 = purified final material post activation with Factor Xa + DTT (5 µΐ); Lane 8 = purified final material post activation with Factor Xa + DTT (10 µΐ); Lane 9 = purified final material post activation with Factor Xa + DTT (20 µΐ); lane 10 = standard molecular weight markers.

Fig. 24 - Expressed / purified CPOP-A product

[0148] Proteins were subjected to SDS-PAGE and then stained with Coomassie blue. The electrophoretic profile shows the purification of a two-chain substance with disulfide bonds of the expected molecular weight of CPOPA. Lane 1 = standard molecular weight markers; Lane 2 = Clean material after initial capture on charged Ni²⁺ a sepharose column; Lane 3 = Factor Xa treated material prior to final capture on charged Ni²⁺ a sepharose column; Lane 4 = Clean material after second capture on charged Ni²⁺ a sepharose column; Lane 5 = purified final material post activation with Factor Xa (5 μΐ); Lane 6 = purified final material post activation with Factor Xa (10 µΐ); Lane 7 = purified final material post activation with Factor Xa (20 μΐ); Lane 8 = purified final material post activation with Factor Xa + DTT (5 µΐ); Lane 9 = purified final material post activation with Factor Xa + DTT (10 µΐ); Lane 10 = purified final material post activation with Factor Xa + DTT (20 µΐ).

Figure 25 - Expressed / purified CPOPv-A product

[0149] Proteins were subjected to SDS-PAGE and then stained with Coomassie blue. The electrophoretic profile shows the purification of the two-chain substance with disulfide bonds with the expected molecular weight of CP0Pv-A. Lane 1 = standard molecular weight markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = Clean material after initial capture on charged Ni²⁺a sepharose column; Lane 4 = Factor Xa treated material prior to final capture on charged Ni²⁺ a sepharose column; Lane 5 = purified final material post activation with Factor Xa (5 μΐ); Lane 6 = purified final material post activation with Factor Xa (10 µΐ); Lane 7 = purified final material post activation with Factor Xa (20 μΐ); Lane 8 = purified final material post activation with Factor Xa + DTT (5 µΐ); Lane 9 = purified final material post activation with Factor Xa + DTT (10 µΐ); Lane 10 = purified final material post activation with Factor Xa + DTT (20 µΐ).

Figure 26 - In vitro cleavage of SNAP-25 in a DRG cell model [0150] Primary dorsal root ganglion (DRG) cultures were exposed to different concentrations of GP0Pv-

And for 24 hours. Cellular proteins were separated by SDS-PAGE and analyzed by Western blotting using an anti-SNAP-25 probe to facilitate evaluation of SNAP25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis.

Fig. 27 - Expressed / purified CPNv-A-FXa-HT (removable his tag)

[0151] Proteins were subjected to SDS-PAGE and then stained with Coomassie blue. The electrophoretic profile shows the purification of the two-chain substance with disulfide bonds with the expected molecular weight of CPNvA-FXa-HT. Lane 1 = standard molecular weight markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = Factor Xa treated material prior to final capture on charged Ni²⁺ a sepharose column; Lane 4 = purified final material post activation with Factor Xa; Lane 5 = purified final material post activation with Factor Xa + DTT.

Figure 28 - In vitro performance of the LC / Anociceptin-H fusion protein_N/ A with variable spacer length, as assessed by a ligand competition assay

[0152] The LC / A-nociceptin-H fusion capacity was assessed by a simple competition test._N/ A with a variable length spacer for binding to the ORL1 receptor. Primary dorsal root ganglion (DRG) cultures were exposed to various concentrations of test material in the presence of 1 nM [³H] -nociceptin. The reduction in the specific binding of the radiolabeled ligand was assessed using a scintillation counter and a graph was drawn comparing the effectiveness of the unlabeled ligand (Tocris nociceptin). The top panel illustrates the displacement characteristics for the GSO, GS20, GS30 and Hx27 spacers, while the bottom panel illustrates the displacement achieved by the GS10, GS15 and GS25 spacer fusion proteins. It can be concluded that the GSO and GS30 spacers are ineffective, and GS10 is ineffective in displacing nociceptin from the ORL1 receptor.

Figure 29 - In vitro efficacy of LC / Anociceptin-HN / A fusion proteins with variable spacer length as assessed by in vitro SNAP-25 cleavage

[0153] Primary dorsal root ganglion (DRG) cultures were exposed to different concentrations of CPN-A (with different spacer lengths) for 24 hours. Cellular proteins were separated by SDS-PAGE and analyzed by Western blotting using an anti-SNAP-25 probe to facilitate evaluation of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. The low efficiency of binding the fusion protein to the GS10 spacer (see Fig. 28) reflects the greater concentration of fusion necessary to obtain intracellular cleavage of SNAP-25. The fusion proteins with the GSO and GS30 spacers were completely ineffective (data not shown). Fusion proteins with spacers GS15, 20 and 25 showed similar efficacy.

Seq id no. (SEQ ID)

[0154]

SEQ ID1

SEQ ID2

SEQ ID3

SEQ ID4

SEQ ID5

SEQ ID6

SEQ ID7

SEQ ID8

SEQ ID9

SEQ ID10

SEQ IDU

SEQ ID12

SEQ ID13

SEQ ID14

SEQ ID15

SEQ ID16

SEQ ID17

SEQ ID18

SEQ ID19

SEQ ID20

SEQ ID21

SEQ ID22

SEQ ID23

SEQ ID24

<td>LC / A DNA sequence DNA sequence of H_N/AND</td>

<td>LC / B DNA sequence</td>

<td>DNA sequence of H_N/ B</td>

<td>LC / C DNA sequence</td>

<td>DNA sequence of H_N/ C</td>

<td>DNA sequence of the CPN-A linker</td>

<td>Linker A DNA sequence</td>

<td>DNA sequence of the N-terminal presentation of the insert</td>

<td>nociceptins</td>

<td>DNA sequence of the CPN-C linker</td>

<td>DNA sequence of the CPBE-A linker</td>

<td>DNA sequence of the CPNvar-A linker</td>

<td>LC / A-CPN-H fusion DNA sequence_n/AND</td>

<td>LC / A-CPN-H fusion protein sequence_n/AND</td>

<td>DNA sequence of the reference fusion molecule</td>

<td>N-LC / AH_n/AND</td>

<td>Protein sequence of a reference molecule</td>

<td>N-LC / AH fusions_n/AND</td>

<td>LC / C-CPN-H fusion DNA sequence_n/ C</td>

<td>LC / C-CPN-H fusion protein sequence_n/ C</td>

<td>LC / C-CPN-H fusion DNA sequence_n/ C (junction A)</td>

<td>LC / C-CPN-H fusion protein sequence_n/ C</td>

<td>(junction A)</td>

<td>LC / A-CPME-H fusion DNA sequence_n/AND</td>

<td>LC / A-CPME-H fusion protein sequence_n/AND</td>

<td>LC / A-CPBE-H fusion DNA sequence_n/AND</td>

<td>LC / A-CPBE-H fusion protein sequence_n/AND</td>

<td>SEQ ID25</td><td>Sequence a</td><td>LC / A-CPNv-H fusion DNA_n/AND</td>

<td>SEQ ID26</td><td>Sequence a</td><td>LC / A-CPNv-H fusion protein_n/AND</td>

<td>SEQ ID27</td><td>Sequence a</td><td>LC / A-CPN [1-11] -HN / A fusion DNA</td>

<td>SEQ ID28</td><td>Sequence a</td><td>LC / A-CPN fusion protein [1-11] -HN / A</td>

<td>SEQ ID29</td><td>Sequence a</td><td>LC / A-CPN fusion DNA [[Y10] 1-11] -HN / A</td>

<td>SEQ ID30</td><td>Sequence a 11] -HN / A</td><td>fusion protein LC / A-CPN [[ΥΙΟ] 1-</td>

<td>SEQ ID31</td><td>Sequence a</td><td>LCIA-CPN fusion DNA [[III] 1-11] -HN / A</td>

<td>SEQ ID32</td><td>Sequence 11] -HN / A</td><td>LC / A-CPN fusion protein [[Y11] 1-</td>

<td>SEQ ID33</td><td>Sequence a</td><td>LC / A-CPN fusion DNA [[Y14] 1-17] -HN / A</td>

<td>SEQ ID34</td><td>Sequence 17] -HN / A</td><td>LC / A-CPN fusion protein [[Y14] 1-</td>

<td>SEQ ID35</td><td>Sequence a</td><td>LC / A-CPN [1-13] -HN / A fusion DNA</td>

<td>SEQ ID36</td><td>Sequence a</td><td>LC / A-CPN fusion protein [1-13] -HN / A</td>

<td>SEQ ID37</td><td>Sequence a</td><td>CPN DNA [1-17]</td>

<td>SEQ ID38</td><td>Sequence a</td><td>protein CPN [1-17]</td>

<td>SEQ ID39</td><td>Sequence a</td><td>CPN DNA [1-11]</td>

<td>SEQ ID40</td><td>Sequence a</td><td>protein CPN [1-11]</td>

<td>SEQ ID41</td><td>Sequence a</td><td>CPN DNA [[Y10] 1-11]</td>

<td>SEQ ID42</td><td>Sequence a</td><td>protein CPN [[Y10] 1-11]</td>

<td>SEQ ID43</td><td>Sequence a</td><td>DNA CPN [[Yll] 1-11]</td>

<td>SEQ ID44</td><td>Sequence a</td><td>protein CPN [[Y11] 1-11]</td>

<td>SEQ ID45</td><td>Sequence a</td><td>CPN DNA [[Y14] 1-17J</td>

<td>SEQ ID46</td><td>Sequence a</td><td>protein CPN [[Y14] 1-17]</td>

<td>SEQ ID47</td><td>Sequence a</td><td>CPN DNA [1-13]</td>

<td>SEQ ID48</td><td>Sequence a</td><td>protein CPN [1-13]</td>

<td>SEQ ID49</td><td>Sequence a N [[R14K15]</td><td>CPNv DNA (also known as 1-17])</td>

<td>SEQ ID50</td><td>Sequence a</td><td>protein CPNv (also known as</td>

N [[R14K15] 1-17])

SEQ ID51

SEQ ID52

SEQ ID53

SEQ ID54

SEQ ID55

SEQ ID56

SEQ ID57

SEQ ID58

SEQ ID59

SEQ ID60

SEQ ID61

SEQ ID62

SEQ ID63

SEQ ID64

SEQ ID65

SEQ ID66

SEQ ID67

SEQ ID68

SEQ ID69

SEQ ID70

SEQ ID71

SEQ ID72

SEQ ID73

SEQ ID74

SEQ ID75

SEQ ID76

SEQ ID77

DNA sequence of the nociceptin-spacer-LC / AH fusion comparison molecule_N/AND

Fusion and nociceptin-spacer-LC / AH protein sequence_N/AND

<td>Sequence a</td><td>GOUT</td><td>CPN-A connector</td><td>GS10</td>

<td>Sequence a</td><td>GOUT</td><td>CPN-A connector</td><td>GS15</td>

<td>Sequence a</td><td>GOUT</td><td>CPN-A connector</td><td>GS25</td>

<td>Sequence a</td><td>GOUT</td><td>CPN-A connector</td><td>GS30</td>

<td>Sequence a</td><td>GOUT</td><td>CPN-A connector</td><td>HX2 7</td>

<td>Sequence a</td><td>GOUT</td><td colspan="2">LC / A-CPN (GS15) -H fusion_n/AND</td>

<td>Sequence a</td><td colspan="2">LC / i fusion protein</td><td>1-CPN (GS15) -H_n/AND</td>

<td>Sequence a</td><td>GOUT</td><td colspan="2">LC / A-CPN (GS25) -H fusion_n/AND</td>

<td>Sequence a</td><td colspan="2">LC / i fusion protein</td><td>1-CPN (GS25) -H_n/AND</td>

DNA sequence of the enterokinase-activated CPNvar-A linker

<td>Sequence a</td><td>LC / A-CPNv (Ek) -H fusion DNA_n/AND</td>

<td>Sequence a Sequence a</td><td>LC / A-CPNv (Ek) -H fusion protein_N/ A DNA of the CPNvar-A linker</td>

<td>Sequence a</td><td>LC / C-CPNv-H fusion DNA_n/ C (updated A)</td>

<td>Sequence A) Sequence Sequence Sequence Sequence Sequence Sequence Sequence Sequence Sequence Sequence</td><td>LC / C-CPNv-H fusion protein_n/ C (act LC / A-CPLE-H fusion DNA_n/ A LC / A-CPLE-H fusion protein_n/ A DNA of LC / A-CPOP-H fusion_n/ A LC / A-CPOP-H fusion protein_n/ A DNA of LC / A-CPOPv-H fusion_n/ A LC / A-CPOPv-H fusion protein_n/ A DNA protease IgA DNA fusion IgA-CPNv-H_N/ A IgA-CPNv-H fusion protein_N/ A DNA of factor Xa-HT</td>

<td>SEQ ID78</td><td>Sequence a</td><td>DNA CPNv-A-Factor Xa-HT</td>

<td>SEQ ID79</td><td>Sequence a</td><td>CPNv-A-FXa-HT fusion protein</td>

<td>SEQ ID80</td><td>Sequence a</td><td>DT translocation domain DNA</td>

<td>SEQ ID81</td><td>Sequence a</td><td>DNA of CPLE-DT-A</td>

<td>SEQ ID82</td><td>Sequence a</td><td>CPLE-DT-A fusion protein</td>

<td>SEQ ID83</td><td>Sequence a</td><td>TeNT LC DNA</td>

<td>SEQ ID84</td><td>Sequence a</td><td>DNA of CPNv-TENT LC</td>

<td>SEQ ID85</td><td>Sequence a</td><td>CPNV-TeNT LC fusion protein</td>

<td>SEQ ID86</td><td>Sequence a</td><td>CPNvar-C linker DNA</td>

<td>SEQ ID87</td><td>Sequence a</td><td>LC / C-CPNv-H fusion DNA_n/ C (updated C)</td>

<td>SEQ ID88</td><td>Sequence (act. C)</td><td>LC / C-CPNv-H fusion protein_n/ C</td>

Examples

Example 1 - Production of LC / A and H backbone clones_n/AND

[0155] The following procedure generates LC and H fragments_N for use as a backbone component for multi-domain fusion expression. The present example is based on the production of a clone based on serotype A (SEQ ID1 and SEQ ID2), although the procedures and methods are equally applicable to other serotypes [as illustrated in the sequence listing for serotype B (SEQ ID3 and SEQ ID4) and serotype C (SEQ ID5 and SEQ ID6)].

Generation of cloning vectors and expression vectors

[0156] The standard cloning vector of choice is pCR 4 (Invitrogen): the reason for this choice is the lack of restriction sequences in the vector and the proximity of sequencing primer sites for easy construct confirmation. The expression vector is based on the pMAL (NEB) expression vector containing the desired restriction sequences within the cluster of cloning sites in the correct orientation for the insert in the construct (BamHI-Sal-Pstl-HindIII). The expression vector fragment was removed to create a non-mobilized plasmid, and various fusion tags were introduced to extend the purification possibilities.

Production of insert - protease (e.g. LC / A) LC / A (SEQ ID1) was prepared in one of two ways:

The DNA sequence is designed by reverse translation of the LC / A amino acid sequence [obtained from publicly available databases such as GenBank (accession no. P10845) or Swissprot (BXA1_CLOBO access site) using various reverse translation programs (e.g. EditSeg best E. coli reverse translation (DNASTAR Inc.) or Backtranslation tool version 2.0 (Entelechon)]. BamBI / SalI recognition sequences are inserted at the 5 'and 3' ends of the sequences, respectively, maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences inserted during reverse translation. Any sequences found, the same as those required by the cloning system, are manually removed from the proposed coding sequence, ensuring that typical E. coli codon usage is maintained. E. coli codon usage is assessed using software such as Graphical Codon Usage Analyzer (Geneart), and% GC content is assessed by reference to published codon usage tables (e.g., GenBank, Edition 143, September 13, 2004). This optimized DNA sequence containing the LC / A open reading frame (ORF) is then produced commercially (for example, produced by Entelechon, Geneart and Sigma-Genosys) and provided in the pCR 4 vector.

[0158] An alternative approach is to use PCR amplification from an existing DNA sequence using the restriction enzyme sequence BamHI and SalI inserted into the 5 'and 3' PCR primers, respectively. Complementary oligonucleotide primers are chemically synthesized by a supplier (e.g. MWG or Sigma-Genosys) such that each pair has the ability to hybridize to opposite strands (3 'ends facing each other), flanking a segment of the target Clostridium DNA, one oligonucleotide for each from two strands of DNA. To produce a PCR product, a pair of short oligonucleotide primers specific to the Clostridial DNA sequence are mixed with the Clostridial DNA template and other reaction components and placed in a device ("PCR device), which can automatically vary the incubation temperature in the reaction tube by setting cycles. at a temperature of about 94 ° C (denaturation), through 55 ° C (oligonucleotide attachment), to 72 ° C (synthesis). Other reagents required to amplify the PCR product include DNA polymerase (e.g., Tag polymerase or

Pfu), each of the four dNTP nucleotides - "DNA building blocks in equimolar amounts (50-200 μΜ) and a buffer appropriate for the enzyme, optimized for Mg concentration²⁺ (0.5-5 mM).

[0159] The amplification product is cloned into pCR 4 using TOPO TA cloning for PCR products using Tag or Zero Blunt TOPO cloning for PCR products using Pfu (both kits are commercially available from Invitrogen). The obtained clone is checked by sequencing. Any additional restriction sequences that do not match the cloning system are then removed using site-directed mutagenesis [for example Quickchange (Stratagene Inc.)].

Producing a translocation insert (e.g. H._N) [0160] Prepare H_N/ A (SEQ ID2) in one of two ways:

The DNA sequence is designed by reverse translation of the H amino acid sequence_N/ A [obtained from publicly available databases such as GenBank (Accession No. P10845) or Swissprot (Access Site BXA1_CLOBO)] using various reverse translation programs [e.g. EditSeg best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool version 2.0 (Entelechon)]. A PstI restriction sequence is added to the N-terminus and Xbal-stop codon-HindIII to the C-terminus, ensuring the correct reading frame is kept. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences inserted during reverse translation. Any sequences found that are common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage. Usage of E codons coli is evaluated by reference to software such as Graphical Codon Usage Analyzer (Geneart), and the% GC content is evaluated by reference to published codon usage tables (e.g., GenBank, Edition 143, September 13, 2004). This optimized DNA sequence is then produced commercially (for example by Entelechon, Geneart and Sigma-Genosys) and provided in the pCR 4 vector.

[0161] An alternative method is to use PCR amplification from an existing DNA sequence by inserting into the primers 5 'and 3', respectively, the sequence for the PstI and Xbal-stop-HindIII restriction enzymes. The PCR amplification is performed as described above. The PCR product is inserted into the pCR 4 vector and checked by sequencing. Any additional restriction sequences that do not match the cloning system are then removed by site-directed mutagenesis [for example using Quickchange (Stratagene Inc.)].

Example 2 - Preparation of LC / Anocyceptin-H fusion protein_N/ A (nociceptin is N-terminal to the H chain_N)

Insertion Preparation: Linker-Nociceptin-Spacer [0162] LC-H linker_N can be designed on the basis of the first principle, using existing linker sequence information as template. The length of the serotype A linker (in this case defined as an interdomain polypeptide region existing between the cysteine ​​residues of the disulfide bridge between LC and H_N) is for example 23 amino acids and its sequence is VRGIITSKTKSLDKGYNKALNDL. In nature, proteolytic activation within this sequence is believed to result in the formation of the H domain_Ncomprising the N-terminus of the ALNDL sequence. This sequence information is publicly available in databases such as GenBank (Accession No. P10845) or Swissprot (Accession BXA1_CLOBO). A factor Xa site, nociceptin and spacer are introduced into this linker, and using various reverse translation programs [e.g., EditSeg best E. coli reverse translation (DNASTAR Inc.) or Backtranslation tool version 2.0 (Entelechon)], the DNA sequence encoding a linker-ligand-spacer region is determined. Restriction sites are then inserted into the DNA sequence and can be arranged in the order of BamAI-Sall linker-protease site-nociceptin-Nhel spacer-Spel-Pstl-Abal-stop codon-HindIII (SEQ ID7). It is important to ensure that the correct reading frame is maintained for the spacer, nociceptin, and restriction sequences, and that the Xbal sequence is not preceded by TC bases, which would result in DAM methylation. The DNA sequence is screened for restriction sequence insertion, and any additional sequences are removed manually from the remainder of the sequence, ensuring common E. coli codon usage. Usage of E codons coli is evaluated by reference to software such as Graphical Codon Usage Analyzer (Geneart), and the% GC content is evaluated by reference to published codon usage tables (e.g., GenBank, Edition 143, September 13, 2004). This optimized DNA sequence is then produced commercially (for example by Entelechon, Geneart and Sigma-Genosys) and provided in the pCR 4 vector.

Generation of LC / A-nociceptin-H fusions_N/AND

[0163] To generate the LC-linkerniciceptin-spacer-H construct_N (SEQ ID13), the pCR 4 vector encoding the linker (SEQ ID7) is cleaved with the restriction enzymes BamHI + SalI. This cleaved vector then serves as a recipient vector to insert and ligate the LC / A DNA (SEQ ID1) cleaved with BamHI + SalI. The resulting plasmid DNA is then cut with the restriction enzymes Pstl + Xbal and serves as a recipient vector for insertion and ligating j and H DNA._N/ A (SEQ ID2) of the cleaved Pstl + Xbal. The final construct contains the LC-linker-nociceptin-spacer-H ORF_N (SEQ ID13) to be transferred into expression vectors for expression, resulting in a fusion protein with the sequence shown in SEQ ID14.

Example 3 - Preparation of a comparative nociceptin-LC / A-HN / A fusion protein molecule (nociceptin is N-terminal to the LC chain)

[0164] The LC / AH backbone was prepared using the method described in Example 2_n/ A, using a synthesized serotype A linker with the addition of a factor Xa site for activation, with the BamHI-Sall linker-protease site-linker-Pst-XbaI-stop codon-HindIII (SEQ ID8). LC / AH backbone_n/ A and the synthesized N-terminal presented nociceptin insert (SEQ ID9) are cleaved with BamHI + HindIII restriction enzymes, gel purified and ligated to form a nociceptin-spacer-LC-linker-H construct_N. The ORF (SEQ ID15) is then excised, using the restriction enzymes Aval + Xbal, for transfer into expression vectors for expression, resulting in a fusion protein having the sequence shown in SEQ ID16.

Example 4 - Preparation of LC / C nociceptin-H fusion protein_N/ C

[0165] By the methods used in Examples 1 and 2, LC / C (SEQ ID5) and H_N/ C (SEQ ID6) and introduces into the BamHI-Sall serotype C linker-protease site-nociceptin-Nhel spacer-Spel-Pstl-Abal-stop codon-HindIII (SEQ ID10). The final construct contains the LC-linker-ciceptin-spacer-H ORF_N (SEQ ID17) to be expressed as a protein with the sequence shown in SEQ ID18.

Example 5 - Preparation of LC / C nociceptin-H fusion protein_N/ C with serotype A activation sequence

[0166] The methods used in Examples 1 and 2 are preparing LC / C (SEQ ID5) and H_N/ C (SEQ ID6) and introduces into the BamHI-Sall serotype A linker-protease site-nociceptin-Nhel spacer-Spel-Pstl-Abal-stop codon-HindIII (SEQ ID7). The final construct contains the LC-linker-ciceptin-spacer-H ORF_N (SEQ ID19) to be expressed as a protein with the sequence shown in SEQ ID20.

Example 6 - Preparation of LC / A-methenkephalin-H fusion protein_N/AND

[0167] Due to the small size of the methenkephalin ligand (it consists of five amino acids), the LC / A-met enkephalin-H fusion_N/ A is generated by site-directed mutagenesis [using e.g. Quickchange (Stratagene Inc.)] using the LC / Anocyceptin-H fusion as template_N/ A (SEQ ID13). Oligonucleotides encoding the YGGFM met-enkephalin peptide are designed, ensuring standard codon usage in E.coli is maintained, and no additional restriction sites are inserted; oligonucleotides are flanked by sequences complementary to the LC / A-nociceptin-H fusion linker region_N/ A (SEQ ID13) on either side of the nociceptin portion. SDM performs well by sequencing; the final construct contains the LC-linker-meth enkephalin-spacer-H ORF_N (SEQ ID21) to be expressed as a protein with the sequence shown in SEQ ID22.

Example 7 - Preparation of LC / Α-β endorphin-H fusion protein_N/AND

[0168] The methods used in Examples 1 and 2 prepared LC / A (SEQ ID1) and H_N/ A (SEQ ID2) and introduces the BamHI-Sall-linker-protease site-β-endorphinNheI-spacer-Spel-Pstl-Xbal-stop codon-HindIII (SEQ IDU) linker into the β-endorphin linker of serotype A. The final construct contains the LC-linker-β endendin-spacer-H ORF_N (SEQ ID23) to be expressed as a protein with the sequence shown in SEQ ID24.

Example 8 - Preparation of LC / A fusion protein - variant no cycepin-H_N/AND

[0169] The methods used in Examples 1 and 2 are prepared for LC / A (SEQ ID1) and Hn / A (SEQ ID2) and insert into the linker of the serotype A nociceptin variant BamHI-Sall-linker-protease site for nt nociceptin-Nhel protease -interruptor-Spel-Pstl-Xbalkodone stop-HindIII (SEQ ID12). The final construct contains the LC-linker-nociceptin variant-spacerH ORF_n (SEQ ID25) to be expressed as a protein with the sequence shown in SEQ ID26.

Example 9 - Purification method for LC / Anocyc ep tyin-H fusion protein_N/AND

[0170] Thaw a Falcon tube containing 25 ml 50 mM HEPES, pH 7.2, 200 mM NaCl, and approximately 10 g BL21 E. coli cell paste. Fill the thawed cell paste to a volume of 80 ml 50 mM HEPES, pH 7.2, 200 mM NaCl and sonicate on ice: 30 seconds on, 30 seconds off - 10 cycles - at 22 microns, keeping the sample cool. Centrifuge the lysed cells at 18,000 rpm and 4 ° C for 30 minutes. Place the supernatant on the loaded 0.1 M NiSO₄ chelating column (20-30 ml column volume is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a 10 and 40 mM imidazole step gradient, wash out the non-specific bound protein and elute the fusion protein with 100 mM imidazole. Dialyze the eluted fusion protein against 5 L 50 mM HEPES pH 7.2, 200 mM NaCl at 4 ° C overnight and measure the OD of the dialyzed fusion protein. Add 1 unit factor Xa per 100 µg fusion protein and incubate static at 25 ° C overnight. Apply to the charged 0.1 M NiSO₄ chelating column (20-30 ml column is sufficient), equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl. Wash the column to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a 10 and 40 mM imidazole step gradient, wash out the non-specific bound protein and elute the fusion protein with 100 mM imidazole. Dialyze the eluted fusion protein against 5 L 50 mM HEPES, pH 7.2, 200 mM NaCl at 4 ° C overnight and concentrate the fusion to 2 mg / ml, take a sample and freeze to -20 ° C. Test the purified protein using OD, BCA, purity analysis, and SNAP-25 tests.

Example 10 - Confirmation of TM agonist activity by determining the release of substance P from neuronal cultures

Materials

EIA for substance P is obtained from R&amp;D Systems, UK.

Methods

[0171] Primary cultures of eDRG neurons are assumed as previously described (Duggan et al., 2002). Release of substance P from the culture is assessed by EIA, essentially as previously described (Duggan et al., 2002). The TM of interest is added to the cultures of neurons (established at least 2 weeks prior to treatment); control cultures are carried out in parallel by adding the vehicle in place of the TM. A stimulated (100 mM KCl) and basal release is obtained, along with the total cell lysate content, of substance P, for both the control and TM-treated cultures. Substance P immunoreactivity is determined using substance P immunoassay kits (Cayman Chemical Company, USA or R&amp;D Systems, UK) according to the manufacturer's instructions.

[0172] The amount of substance P released by neurons in the presence of the TM of interest is compared with the release obtained in the presence and absence of 100 mM KCl. The stimulation of substance P release by the TM of interest over the baseline release means that the TM of interest is an "agonist ligand as defined herein. If desired, the stimulation of substance P release by the TM of interest can be compared with a standard substance P release curve obtained using the natural ORL-1 receptor ligand nociceptin (Tocris).

Example 11 - Confirmation of ORL1 receptor activation by assaying forskolin stimulated cAMP production

[0173] Confirmation that the TM of interest acts through the ORL1 receptor is provided by the following assay in which the ability of the TM to inhibit forskolin stimulated cAMP production is assessed.

Materials

[0174] [³H] adenine and [¹⁴C] cAMP was obtained from GE Healthcare.

Methods

[0175] The test is performed essentially as previously described by Meunier et al. [Isolation and structure of the endogenous agonist of opioid receptor-like ORLi receptor. Nature 377: 532-535,1995] in intact, transfected CHO cells, plated in 24-well plastic plates.

[0176] [³H] adenine (1.0 μCi) in 0.4 ml of culture medium. Cells remain at 37 ° C for 2 h, which allows adenine to be incorporated into the intracellular ATP pool. After 2 h, the cells are washed once with the incubation buffer containing: 130 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂AFTER₄, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 10 mM glucose, 1 mg / ml bovine serum albumin and 25 mM HEPES, pH 7.4, and replaced with a buffer containing forskolin (10 µM) and isobutylmethylxanthine (50 µL) with or without the TM of interest. After 10 min, the medium is aspirated and replaced with 0.5 ml of 0.2 M HCl. Approximately 1000 cpm is added to each well [¹⁴C] cAMP and is used as an internal standard. The contents of the wells are then transferred to columns containing 0.65 g of dry alumina powder. The columns are eluted with 4 ml of 5 mM HCl, 0.5 ml of 0.1 M ammonium acetate, and then with two additional ml of ammonium acetate. The final eluate is collected in scintillation vials and counted¹⁴C and tritium. The resulting amounts are corrected for recovery [¹⁴C] cAMP. TMs that are agonists of the ORL1 receptor cause a reduction in the level of cAMP produced in response to forskolin.

Example 12 - Confirmation of ORL1 receptor activation using a functional GTP? S binding assay

Confirmation that a given TM is acting through the ORL1 receptor is also provided by the following assay, a functional GTP? S binding assay.

Materials

[0178] [³⁵S] GTPyS is obtained from GE Healthcare. Wheat Germ Agglutinin (SPA) coated beads are obtained from GE Healthcare.

Methods

[0179] This test is performed essentially as described by Traynor and Nahorski [Modulation by μ-opioid agonists of guanosine-5-0- (3- [³⁵S] thio) triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Moth. Pharmacol. 47: 848-854,1995].

[0180] Cells are scraped from tissue culture plates and placed in 20mM HEPES, 1mM ethylenediaminetetraacetic acid followed by centrifugation at 500 xg for 10 min. The cells are then resuspended in this buffer and homogenized in the Polytron Homogenizer.

[0181] The homogenate is centrifuged at 27,000 xg for 15 min and the pellet is resuspended in buffer A containing: 20 mM HEPES, 10 mM MgCl₂, 100 mM NaCl, pH 7.4. The suspension is centrifuged again at 20,000 xg and resuspended in buffer A. For the membrane binding assay (8-15 μg of protein) is incubated with [³⁵S] GTP S (50 µM), GDP (10 µΜ), with and without the TM of interest, in a total volume of 1.0 ml, for 60 min at 25 ° C. Samples are filtered on glass fiber filters and counted as described for the binding assays.

Example 13 - Preparation of LC / Anocyceptin-H fusion protein_N/ A (nociceptin is N-terminal to the H chain_N)

[0182] The linker-nociceptin-spacer insert is prepared as described in Example 2.

Generation of LC / A-nociceptin-H fusions_N/AND

[0183] To generate the LC-linkerniciceptin-spacer-H construct_N (SEQ ID13), the pCR 4 vector encoding the linker (SEQ ID7) is cleaved with the restriction enzymes BamHI + SalI. This cleaved vector then serves as a recipient to insert and ligate LC / A DNA (SEQ ID1), also cleaved with BamHI + SalI. The resulting plasmid DNA is then cut with the restriction enzymes BamHI + HindIII and the LC / linker A fragment is inserted into a similarly cut vector containing a unique cluster of cloning sites for BamHI, SalI, PstI, and HindIII, such as the pMAL (NEB) vector. DNA of H._n/ A (SEQ ID2) is then cleaved with Pstl + HindIII restriction enzymes and inserted into a similarly cleaved pMAL-LC / linker A construct. The final construct contains the LC-linkernocyceptin-spacer-H ORF_N (SEQ ID13) to be expressed as a protein with the sequence shown in SEQ ID14.

Example 14 - Preparation of the nociceptin-LC / AH fusion protein reference molecule_N/ A (nociceptin is N-terminally to the LC chain) [0184] To generate the nociceptin-spacer-LC / AH construct_N/ A, a serotype A linker with the addition of a factor Xa activation site is synthesized with the BamHI-Sall-linker-protease site linker-PstI-Xbal-stop codon-HindIII (SEQ ID8) as described in example 13. Vector pCR 4 encoding linker is cleaved with BamHI + SalI restriction enzymes. This cleaved vector then serves as a recipient for insertion and ligation of LC / A DNA (SEQ ID1), also cleaved with BamHI + SalI. The resulting plasmid DNA is then cut with the restriction enzymes BamHI + HindIII and the LC / linker A fragment is inserted into a similarly cut vector containing the synthesized N-terminal nociceptin insert (SEQ ID9). This construct is then cut with Aval + HindIII and inserted into an expression vector such as the plasmid pMAL (NEB). DNA of H._N/ A (SEQ ID2) is then cleaved with the restriction enzymes Pstl + HindIII and inserted into the similarly cleaved construct pMAL-nociceptin-LC / linker A. The final construct contains the nociceptin-spacer-LC / AH ORF_N/ A (SEQ ID51) to be expressed as a protein with the sequence shown in SEQ ID52.

Example 15 - Generation and purification of the LC / A-nociceptin-H family of fusion proteins_N/ A with a variable length cutout

[0185] Using the same strategy as in Example 2, a series of DNA linkers were generated, encoding nociceptin and variable spacer content. Using various reverse translation programs [for example EditSeg best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool version 2.0 (Entelechon)], a DNA sequence encoding a linker-ligand-spacer region was determined. Restriction sites are then inserted into the DNA sequence and can be arranged as BamHI-SalI-linker-protease site-nociceptin-Nhel-spacer-Spel-Pst-Xbalkodone stop-HindIII (SEQ ID53 to SEQ ID57). It is important to ensure the correct reading frame of the spacer, nociceptin, and restriction sequences is maintained, and to ensure that the Xbal sequence is not preceded by TC bases that would cause DAM methylation. The DNA sequence is screened for restriction sequence insertion and any additional sequences are removed manually from the remainder of the sequence, ensuring that typical E. coli codon usage is maintained. Usage of E codons coli is evaluated by reference to software such as Graphical Codon Usage Analyzer (Geneart); % GC content and codon usage are evaluated by reference to published codon usage tables (e.g., GenBank, Edition 143, September 13, 2004). This optimized DNA sequence is then produced commercially (for example by Entelechon, Geneart and Sigma-Genosys) and provided in the pCR 4 vector.

[0186] The spacers produced included the following:

Table 1

<td>Code</td><td>Linker protein sequence</td><td>SEQ ID DNA of the linker</td>

<td>GS10</td><td>ALAGGGGSALVLQ</td><td> 53</td>

<td>GS15</td><td>ALAGGGGSGGGGSALVLQ</td><td> 54</td>

<td>GS25</td><td>ALAGGGGSGGGGSGGGGSGGGGSALVLQ</td><td> 55</td>

<td>GS30</td><td>ALAGGGGSGGGGSGGGGSGGGGSGGGGSALVLQ</td><td> 56</td>

<td>HX27</td><td>ALAAEAAAKEAAAKEAAAKAGGGGSALVLQ</td><td> 57</td>

[0187] As an example, in order to generate an LC / A-CPN (GS15) -H fusion construct_N/ A (SEQ ID58), the pCR 4 vector encoding the linker (SEQ ID54) is cleaved with the restriction enzymes BamHI + SalI. This cleaved vector then serves as a recipient vector for insertion and ligation of the LC / A DNA (SEQ ID1), also cleaved with BamHI + SalI. The resulting plasmid DNA is then cut with the restriction enzymes BamHI + HindIII and the LC / linker A fragment is inserted into a similarly cut vector containing a unique cluster of cloning sites for BamHI, SalI, PstI, and HindIII, such as the pMAL (NEB) vector. DNA of H._n/ A (SEQ ID2) is then cleaved with Pstl + HindIII restriction enzymes and inserted into the similarly cleaved pMAL-LC / linker A construct. The final construct contains the LC / A-CPN (GS15) -H ORF_N/ A (SEQ ID58) to be expressed as a protein with the sequence shown in SEQ ID59.

[0188] As a further example, in order to generate an LC / A-CPN (GS25) -H fusion construct_N/ A (SEQ ID60), the pCR 4 vector encoding the linker (SEQ ID55) is cleaved with the restriction enzymes BamHI + SalI. This cleaved vector then serves as a recipient vector for insertion and ligation of the LC / A DNA (SEQ ID1) cleaved with BamHI + SalI. The resulting plasmid DNA is then cut with the restriction enzymes BamHI + HindIII and the LC / linker A fragment is inserted into a similarly cut vector containing a unique cluster of cloning sites for BamHI, SalI, PstI, and HindIII, such as the pMAL (NEB) vector. DNA of H._n/ A (SEQ ID2) is then cleaved with Pstl + HindIII restriction enzymes and inserted into the similarly cleaved pMAL-LC / linker A construct. The final construct contains the LC / A-CPN (GS25) -H ORF_N/ A (SEQ ID60) to be expressed as a protein with the sequence shown in SEQ ID61.

[0189] LC / A-CPNH fusion variants are similarly constructed_n/ A, consisting of GS10, GS30 and ΗΧ27. Using the purification methodology described in Example 9, the fusion protein is purified from E. coli cell paste. Fig. 9 shows the purified product obtained with

LC / A-CPN (GS10) -H_N/ A, LC / A-CPN (GS15) -H_n/ A, LC / ACPN (GS25) -H_n/ A, LC / A-CPN (GS30) -H_n/ A and LC / A-CPN (ΗΧ27) H_n/AND.

Example 16 - In Vitro Evaluation of LC / Anocyceptin-H Fusion Performance_N/AND

[0190] The fusion protein produced according to pExamples 2 and 9 was assessed in the eDRG neuron model.

[0191] Assays for inhibition of substance P release and SNAP-25 cleavage have been previously reported (Duggan et al., 2002, J. Biol. Chem., 277, 34846-34852). Briefly, dorsal root ganglion neurons from 15-day-old fetuses of Sprague-Dawley rats are harvested and the split cells are plated into 24-well Matrigel coated plates at a density of 1 × 10⁶cells / well. One day after seeding, cells are treated with 10 μΜ cytosine-3-D-arabinofuranoside for 48 h. Cells are maintained in Dulbecco's minimal essential medium supplemented with 5% heat-inactivated fetal bovine serum, 5 mM L-glutamine, 0.6% D- glucose, 2% B27 supplement and 100 ng / ml 2.5S murine nerve growth factor. The cultures are maintained for 2 weeks at 37 ° C in 95% air / 5% CO₂ before adding test materials.

[0192] The release of substance P from e-DRG is assessed in an enzyme-linked immunosorbent assay. Briefly, eDRG cells are washed twice with a low-potassium balanced salt solution (BSS: 5 mM KC1, 137 mM NaCl, 1.2 mM MgCl₂, 5 mM glucose, 0.44 mM KH₂AFTER₄, 20 mM HEPES, pH 7.4, 2 mM CaCl₂). Baseline samples are obtained by incubating each well for 5 min with 1 ml of low potassium BSS. After removal of this buffer, cells are stimulated to release by incubation with 1 ml of high potassium buffer (BSS as above with the modification that 100 mM KC1 isotonic balanced NaCl is added) for 5 min. Prior to substance P testing, all samples are transferred to tubes on ice. Total cell lysates are prepared by adding 250 µΐ 2 M acetic acid / 0.1% trifluoroacetic acid to achieve cell lysis, centrifugation evaporation and resuspension in 500 µ assay buffer. Diluted samples are assessed for substance P content. Substance P immunoreactivity is determined using substance P enzyme immunoassay kits (Cayman Chemical Company or R&amp;D Systems) according to the manufacturer's instructions. Substance P is expressed in pg / ml against a standard parallel curve for substance P.

[0193] SDS-PAGE and Western blotting were performed using standard protocols (Novex). SNAP-25 proteins were separated on a 12% Tris / glycine polyacrylamide gel (Novex) and then transferred to a nitrocellulose membrane. The membranes were incubated with a monoclonal antibody (SMI81) recognizing cleaved and intact SNAP-25. Specific binding was visualized using peroxidase-conjugated secondary antibodies and a chemiluminescent detection system. Cleavage of SNAP-25 was quantified by scanning densitometry (Molecular Dynamics Personal SI, ImageQuant data analysis software). The percentage of SNAP-25 cutting was calculated using the formula:

(SNAP-25 cut / (SNAP-25 cut + intact)) x100.

[0194] After exposure of eDRG neurons to LC / Anocyceptin-H fusion_N/ A (referred to as CPN-A), both inhibition of substance P release and cleavage of SNAP-25 were observed (Figure 10). After 24 h exposure to the fusion, 50% of maximal SNAP-25 cleavage is achieved at a fusion concentration of 6.3 ± 2.5 nM.

[0195] The fusion effect is also assessed at specific time points after 16 h eDRG exposure to CPN-A. Figure 11 illustrates the long duration of action of the CPN-A fusion protein: measurable activity is still observed at 28 days post exposure.

Example 17 - In Vitro Evaluation of LC / Avariant Nociceptin-H Fusion Efficiency_N/AND

[0196] The fusion protein prepared according to Examples 8 and 9 was assessed on eDRG neurons by the method described in Example 16.

[0197] After exposure of eDRG neurons to LC / Nociceptin-H avariant fusion_N/ A (referred to as CPNv-A), both inhibition of substance P release and cleavage of SNAP-25 are observed. After 24 h of exposure to the fusion, 50% of maximal SNAP-25 cleavage is achieved at a fusion concentration of 1.4 0.4 nM (Fig. 12).

[0198] The fusion effect is also assessed at specific time points after 16 h of eDRG exposure to CPN-A. Figure 13 illustrates the long duration of action of the CPN-A fusion protein: measurable activity is still observed at 24 days post exposure.

[0199] The binding capacity of the CPNv-A fusion protein is also assessed in comparison to the CPN-A fusion. Fig. 14 illustrates the results of the competition assay in which binding efficiency at the ORL1 receptor is determined. It has been proven that CPNv-A displaces [³H] -nociceptin, confirming that the receptor can be accessed, with the ligand in a central display format.

Example 18 - Preparation of LC / A-Nociceptin-H variant fusion protein_N/ A, activated by treatment with enterokinase

[0200] The methods used in Examples 1 and 2 were prepared by LC / A (SEQ ID1) and H_N/ A (SEQ ID2) and introduces into the linker the nociceptin serotype A variant of the system BamHI-Sall-linker-enterokinase protease site-nociceptin variant-NheI-spacer-SpelPst-XbaI-stop codon-HindIII (SEQ ID62). The final construct contains the LC-linker-nociceptin variant-spacer-H ORF sequences_N (SEQ ID63) to be expressed as a protein with the sequence shown in SEQ ID64. The fusion protein is referred to as CPNv (Ek) -A. Figure 15 shows the purification of CPNv (Ek) -A from E. coli according to the methods used in Example 9, but using 0.00064 µg per 100 µg of fusion protein to activate enterokinase.

Example 19 - In Vitro Evaluation of LC / Avariant Nociceptin-H Fusion Efficiency_N/ A, activated by treatment with enterokinase

[0201] CPNv (Ek) -A produced in Example 18 is obtained in a purified form and used in the eDRG cell model to evaluate SNAP-25 cleavage (using the methodology in Example 16). Fig. 16 illustrates the cut

SNAP-25 after 24 h eDRG exposure to CPNv (Ek) -A. The cutting performance is found to be similar to the material cut with factor Xa as noted in Example 17.

Example 20 - Preparation of LC / C-Nociceptin-H variant fusion protein_N/ C with factor Xa activation linker derived from serotype A

[0202] The methods used in Example 4 prepared LC / C (SEQ ID5) and H_N/ C (SEQ ID6) and inserts it into the linker of the nociceptin serotype A variant of the BamHI-Sal-linker-nociceptin variant-Nhel-spacer -SpeI-Pst-XbaI-stop codon-HindIII (SEQ ID65). The final construct contains the LC-linker-nociceptin variant-spacer-H ORF sequences_N (SEQ ID66) to be expressed as a protein with the sequence shown in SEQ ID67. The fusion protein is referred to as CPNv-C (act. A). Fig. 17 shows the purification of CPNv-C (act A) from E. coli by the methods used in Example 9.

Example 21 - In vitro evaluation of the efficacy of LC / C-variant nociceptin-H fusion protein_N/ C

[0203] By the methods used in Example 9, CPNv-C (act A) prepared in Example 20 is obtained in a purified form and used in the eDRG cell model to evaluate SNAP-25 cleavage (using the methodology in Example 16). After 24 h exposure to the fusion, 50% of maximal syntaxin cleavage is achieved at a fusion concentration of 3.1 ± 2.0 nM. Figure 18 shows the cleavage of syntaxin after 24 h of e-DRG exposure to CPNv-C (act A).

Example 22 - In Vitro Evaluation of LC / Anocyceptin-HN / A Fusion Efficiency

[0204] The ability of LC / A-nociceptin-H fusion was assessed_N/ A (CPN / A) to inhibit the acute, capsaicin-induced mechanical allodynia following subcutaneous injection of capsaicin into the plantar of the rat hind paw. The paw withdrawal frequency (PWF%) was assessed in test animals in response to a series of stimuli of 10 g von Frey filaments (10 stimuli x 3 samples) prior to enrollment, after subcutaneous administration of CPN / A, but before capsaicin, and after exposure to capsaicin after CPN / A injection (mean of response after 15 'and 30'). Capsaicin exposure is achieved by injecting 10 μΐ 0.3% solution. Sample dilutions are made in 0.5% BSA / saline. Fig. 19 shows the removal of mechanical allodynia achieved by pretreating the animals with a specified concentration range of LC / Anocyceptin-HN / A fusion.

[0205] The ability of the LC / A-nociceptin-HN / A (CPN / A) fusion to inhibit streptozotocin (STZ) induced mechanical (touch responsive) allodynia in rats was assessed. STZ-induced mechanical allodynia in rats is achieved by injection of streptozotocin (ip or iv), which destroys pancreatic β-cells leading to loss of insulin production accompanied by metabolic stress (hyperglycemia and hyperlipidemia). STZ therefore induces type I diabetes. In addition, STZ administration leads to the progressive development of neuropathy serving as a model of chronic pain, with hypersensitivity to pain and allodynia, which may reflect the symptoms seen in people with diabetes (diabetic peripheral neuropathy).

[0206] Male Sprague-Dawley rats (250-300 g) are administered 65 mg / kg STZ in citrate buffer (iv) and blood glucose and lipids are measured weekly to determine model readiness. The Paw Withdrawal Threshold (PWT) is measured in response to a series of von Frey filament stimuli for a specified period of time. Allodynia is considered to have developed when the PWT on two consecutive test days (1 week apart) is less than 6 g on the scale. At this point, the rats are randomly assigned to the saline group (negative efficacy control), the gabapentin group (positive efficacy control), or the test group (CPN / A). Test materials (20-25 µΐ) are injected subcutaneously as a single injection (except gabapentin) and the PWT is measured on day 1 after treatment and periodically thereafter over a period of 2 weeks. Gabapentin (30 mg / kg ip, volume of each injection: 3 ml / kg) is injected daily, 2 hours before the start of the PWT tests. Fig. 20 illustrates the resolution of allodynia obtained by pretreatment of animals with 750 ng CPN / A. Data were acquired for 2 weeks after a single injection of CPN / A.

Example 23 - In Vitro Evaluation of LC / Avariant Nociceptin-H Fusion Efficiency_N/AND

[0207] LC / A-Nociceptin-H variant fusion ability was assessed_N/ A (CPNv / A) to inhibit capsaicin-induced mechanical allodynia

Following subcutaneous injection into the plantar of the rat's hind paw. Test animals are assessed for paw withdrawal frequency (PWF%) in response to a series of stimuli with 10 g von Frey filaments (10 stimuli x 3 trials) prior to enrollment (pre-treatment), after subcutaneous injection of CPNv / A to the footbed, but before capsaicin administration (before CAP), and after capsaicin exposure after CPNv / A injection (mean response after 15 'and 30'; CAP). Capsaicin exposure is achieved by injecting 10 μΐ 0.3% solution. Sample dilutions are made in 0.5% BSA / saline.

[0208] Figure 21 shows resolution of allodynia obtained by pretreatment of LC / Avariant Nociceptin-H fusion animals._N/ A (at various concentrations) compared to the severity obtained by adding the LC / A-nociceptin-H fusion_N/AND. These data are expressed as the normalized paw withdrawal rate differential, in which the difference between the peak response (after capsaicin) and the baseline response (before capsaicin) is presented as a percentage. Based on this analysis, it can be concluded that CPNv / A is more potent than CPN / A, because a lower dose of CPNv / A is sufficient to achieve a similar analgesic effect as the effect observed with CPN / A.

Example 24 - Preparation of LC / A-leu enkephalin-H fusion protein_N/AND

[0209] Due to the small size of the leuenkephalin ligand (it consists of five amino acids), the LC / A-leu enkephalin-H fusion_N/ A is produced by site-directed mutagenesis using for example Quickchange (Stratagene Inc.)] using the LC / Anociceptin-H fusion as template_N/ A (SEQ ID13). Oligonucleotides encoding the YGGFL leu-enkephalin peptide are designed, ensuring that standard E. coli codon usage is retained, and no additional restriction sites are inserted; oligonucleotides are flanked by sequences complementary to the LC / A-nociceptin-H fusion linker region_N/ A (SEQ ID13) on each side of the nociceptin portion. SDM performs well by sequencing; final construct containing the LC-linker-leu enkephalin-spacer-H ORF_N (SEQ ID68) to be expressed as a protein with the sequence shown in SEQ ID69. The fusion protein is referred to as CPLE-A. Figure 22 shows the purification of E. coli CPLE-A by the methods used in Example 9.

Example 25 - Expression and purification of LC / A-beta-endorphin-H fusion protein_N/AND

[0210] The methods used in Example 9, using the LC / A-beta-endorphin-H fusion protein_N/ A (codenamed CPBE-A) prepared in Example 7, CPBE-A from E. coli is purified. Figure 23 shows the purified protein analyzed by SDS-PAGE.

Example 26 - Production of LC / A-nociceptin-H mutant fusion protein_N/AND

[0211] Due to the single amino acid modification necessary for mutation of the nociceptin sequence at position 1 from Phe to Tyr, the LC / A-nociceptin mutant fusion

H._n/ A is produced by site-directed mutagenesis [using e.g. Quickchange (Stratagene Inc.)] using the LC / A-nociceptin-H fusion as template_N/ A (SEQ ID13). Oligonucleotides encoding tyrosine at position 1 of the nociceptin sequence are designed, ensuring standard E. coli codon usage is maintained and no additional restriction sites are inserted; oligonucleotides are flanked by sequences complementary to the LC / A-nociceptin-H fusion linker region_N/ A (SEQ ID13) on each side of the nociceptin portion. SDM performs well by sequencing; final construct containing LC / A fusion ORF-nociceptin mutant-spacer-H_N/ A (SEQ ID70) to be expressed as a protein with the sequence shown in SEQ ID71. The fusion protein is referred to as CPOP-A. Fig. 24 shows the purification of E. coli CPOPA by the methods used in Example 9.

Example 27 - Production and evaluation of LC / A-mutant nociceptin-H fusion protein_N/AND

[0212] Due to the single amino acid modification necessary for mutation of the nociceptin sequence at position 1 from Phe to Tyr, the LC / A-variant nociceptin-H mutant fusion_N/ A is produced by site-directed mutagenesis [using e.g. Quickchange (Stratagene Inc.)] using the LC / Avariant Nociceptin-H fusion as template_N/ A (SEQ ID25). Oligonucleotides encoding tyrosine at position 1 of the nociceptin sequence are designed, ensuring standard E. coli codon usage is maintained and no additional restriction sites are inserted;

oligonucleotides are flanked by sequences complementary to the LC / Avariant Nociceptin-H fusion linker region_N/ A (SEQ ID25) on each side of the nociceptin portion. SDM performs well by sequencing; final construct containing LC / A fusion ORF-nociceptin mutant-spacer-H_N/ A (SEQ ID72) to be expressed as a protein with the sequence shown in SEQ ID73. The fusion protein is referred to as CPOPv-A. Fig. 25 shows the purification of E. coli CPOPv-A by the methods used in Example 9.

[0213] Using the methodology described in Example 16, CPOPv-A is assessed for its ability to cleave SNAP-25 in the eDRG cell model. Figure 26 shows that CPOPv-A is capable of cleaving SNAP-25 in the eDRG model, thereby achieving a 50% cleavage of maximal SNAP-25 after exposing the cells to approximately 5.9 nM of fusion for 24 h.

Example 28 - Production of the fusion protein IgA protease-Nociceptin-H variant_N/AND

[0214] The amino acid sequence of the IgA protease was obtained from publicly available databases such as GenBank (Accession No. P09790). Information on the structure of the N. gonorrhoeae IgA protease gene is available in the literature (Pohlner et al., Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease, Nature, 1987, 325 (6103), 458-62). Using the Backtranslation tool version 2.0 (Entelechon), the DNA sequence encoding the IgA protease modified for expression in E. coli. The recognition sequence for BamHI and a codon encoding the amino acid cysteine ​​were inserted at the 5 'end of the IgA DNA, and the SalI recognition sequence was inserted at the 3' end. The DNA sequence was screened with MapDraw (DNASTAR Inc.) for restriction enzyme cleavage sequences inserted during reverse translation. Any cleavage sequences that are the same as those necessary for cloning are removed manually from the proposed coding sequence, ensuring that typical E. coli codon usage is maintained. Codon usage of E. coli was assessed with a Graphical Codon Usage Analyzer (Geneart); % GC content and codon usage were assessed by reference to published codon usage tables. This optimized DNA sequence (SEQ ID74), containing the IgA open reading frame (ORF), is then produced on a commercial scale.

[0215] The IgA (SEQ ID74) is inserted into the LC ORF-linker nociceptin variant-spacer-H_N (SEQ ID25), using the restriction enzymes BamHI and SalI to replace the LC with DNA for the IgA protease. The final construct contains an IgA-linker-nociceptin variant-spacerHN ORF (SEQ ID75) for expression as a protein with the sequence shown in SEQ ID76.

Example 29 - Production and evaluation of a nociceptin targeting endopeptidase fusion protein with a removable histidine purification tag. DNA was generated encoding the removable factor Xa his tag (his6); it is evident that alternative protease sites such as enterokinase sites and alternative purification tags such as longer histidine tags can also be used. Using various reverse translation programs [e.g. EditSeg best E. coli reverse translation (DNASTAR Inc.) or Backtranslation tool version 2.0 (Entelechon)], the DNA sequence encoding the removable factor Xa region of the his tag is determined. Restriction sites are then inserted into the DNA sequence and can be arranged as Nhel-Linker-Spel-PstlH_N/ A-XbaI-LEIEGRSGHHHHHH Stop-HindIII codon (SEQ ID77). The DNA sequence is screened for the inserted restriction sequence; any additional sequences are removed manually from the remainder of the coding sequence, ensuring that typical E. coli codon usage is maintained. Usage of E codons coli is evaluated by reference to software such as Graphical Codon Usage Analyzer (Geneart), and the% GC content is evaluated by reference to published codon usage tables (e.g., GenBank, Edition 143, September 13, 2004). This optimized DNA sequence is then produced commercially (for example by Entelechon, Geneart and Sigma-Genosys) and provided in the pCR 4 vector. To generate CPNvA-FXa-HT (SEQ ID78 removable his tag construct), the pCR 4 vector encoding the removable hist tag is cut with NheI and HindIII. The Nhel-HindIII fragment is then inserted into the LC / A-CPNv-H vector_n/ A (SEQ ID25), which was also cut by Nhel and Hindlll. The final construct contains the LC ORF / A linker-nociceptin variant-spacer-H sequences_N-FXa His-HindIII tag (SEQ ID78) to be expressed as a protein of the sequence illustrated in SEQ ID79. Fig. 27 shows the purification of CPNv-A-FXa-HT from E. coli by the methods used in Example 9.

Example 30 - Production of a leuenkephalin-targeted endopeptidase fusion protein containing a diphtheria toxin derived translocation domain

[0217] A DNA sequence is designed by reverse translation of the amino acid sequence of a diphtheria toxin translocation domain (obtained from publicly available databases such as GenBank (Accession No. 1XDTT) using various reverse translation programs [for example EditSeg best E. coli reverse translation ((for example) DNASTAR Inc.) or Backtranslation tool version 2.0 (Entelechon)]. Restriction sites are then inserted into the DNA sequence and can be arranged as NheI-linker-Spel-Pstl-diphtheria translocation domain Xbal-stop codon-HindIII (SEQ ID80). Recognition sequences for Pstl / Xbal are inserted at the 5 'and 3' ends of the translocation domain, respectively, with due regard to keeping the correct reading frame in the sequence. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences inserted during reverse translation. Any cleavage sequences found common to those required by the cloning system are removed manually from the remainder of the coding sequence ensuring that typical E. coli codon usage is maintained. Usage of E codons coli is evaluated by reference to software such as Graphical Codon Usage Analyzer (Geneart) and the% GC content is evaluated by reference to published codon usage tables (e.g., GenBank, Edition 143, September 13, 2004). This optimized DNA sequence containing a diphtheria translocation domain is then produced commercially as NheI-Linker-Spel-Pstl-diphtheria translocation domain-XbaI-stop codon-HindIII (for example, manufactured by Entelechon, Geneart and Sigma-Genosys) and provides the pCR 4 vector (Invitrogen). The pCR 4 vector encoding the diphtheria translocation domain is cut with NheI and Xbal. The Nhel-Xbal fragment is then inserted into the LC / A-CPLE-HN / A vector (SEQ ID68), which was also cleaved by NheI and Xbal. The final construct contains the ORF / A-leu-enkephalin-spacer-diphtheria translocation domain (SEQ ID81) ORF sequences for expression as a protein with the sequence shown in SEQ ID82.

Example 31 - Generation of a nociceptin variant endopeptidase fusion protein containing a tetanus toxin-derived LC domain [0218] A DNA sequence is designed by reverse translation of the tetanus LC toxin amino acid sequence (obtained from publicly available databases such as GenBank (Accession No. Χ04436) using various reverse translation programs [e.g., EditSeg best E. coli reverse translation (DNASTAR Inc.) or Backtranslation tool version 2.0 (Entelechon)]. Recognition sequences for BamHI / Sali are inserted at the 5 'and 3' ends, respectively, with respect to keeping the correct reading frame (SEQ ID83). The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences inserted during reverse translation. Any cleavage sequences found common to those required by the cloning system are removed manually from the remaining coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software such as Graphical Codon Usage Analyzer (Geneart), and% GC content is assessed by reference to published codon usage tables (e.g., GenBank, Edition 143, September 13, 2004). This optimized DNA sequence, containing the LC tetanus toxin open reading frame (ORF), is then produced on a commercial scale (for example by Entelechon, Geneart or Sigma-Genosys) and delivered in the pCR 4 vector (Invitrogen). The pCR 4 vector encoding TeNT LC is cut with BamHI and SalI. The BamHISall fragment is then inserted into the LC / A-CPNv-H vector_n/ A (SEQ ID25), which was also cut by BamHI and Sali. The final construct contains the TeNT LC ORF sequences-linker-nociceptin variant-spacer-H_N (SEQ ID84) to be expressed as a protein with the sequence shown in SEQ ID85.

100

Example 32 - Preparation of LC / C-Nociceptin-H variant fusion protein_N/ C with a native serotype C linker, amenable to cleavage with factor Xa

[0219] According to the methods used in Example 4, LC / C (SEQ ID5) and H_n/ C (SEQ ID6) is prepared and inserted into the connector of the nociceptin serotype C variant BamHI-Sali-linker-nociceptin variant-Nhel-spacer SpeI-PstI-XbaI-stop codon-HindIII (SEQ ID86). The final construct contains the sequences of the LC ORF-linker nociceptin variant-spacer-H_N (SEQ ID87) to be expressed as a protein with the sequence shown in SEQ ID88. The fusion protein is referred to as CPNv-C (act. C).

SEQUENCE LIST

[0220] &lt;110&gt; Health Protection Agency Allergan, Inc.

Chaddock, John Foster, Keith Marks, Philip

Stancombe, Patrick Aoki, K Roger

101

Francis, Joseph

Steward, Lance &lt;120&gt; Fusion Proteins &lt;130&gt; P26885WO / MRM &lt;150&gt; GB0426394.3 &lt;151&gt; 2004-12-01 &lt;160&gt; 88 &lt;170&gt; Patentln version 3.3 &lt;210&gt; 1 &lt;211&gt; 1302 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; synthetic &lt;400&gt; 1

<td>ggatccatgg</td><td>agttcgttaa</td><td>caaacagttc</td><td>aactataaag</td><td>acccagttaa</td><td>cggtgttgac</td><td> 60</td>

<td>attgcttaca</td><td>tcaaaatccc</td><td>gaacgctggc</td><td>cagatgcagc</td><td>cggtaaaggc</td><td>attcaaaatc</td><td> 120</td>

<td>cacaacaaaa</td><td>tctgggttat</td><td>cccggaacgt</td><td>gataccttta</td><td>ctaacccgga</td><td>agaaggtgac</td><td>1B0</td>

<td>ctgaacccgc</td><td>caccggaagc</td><td>gaaacaggtg</td><td>ccggCatctt</td><td>actatgactc</td><td>cacctacctg</td><td> 240</td>

<td>tctaccgata</td><td>acgaaaagga</td><td>caactacctg</td><td>aaaggtgtta</td><td>ctaaactgtt</td><td>cgagcgtatt</td><td> 3 00</td>

<td>tactecaccg</td><td>acctgggccg</td><td>tatgctgctg</td><td>actagcatcg</td><td>ttcgcggtat</td><td>cccgttctgg</td><td> 360</td>

<td>ggcggttcta</td><td>ccatcgatac</td><td>cgaactgaaa</td><td>gtaatcgaca</td><td>ctaactgcat</td><td>caacgttatt</td><td> 420</td>

<td>cagccggacg</td><td>gttcctatcg</td><td>ttccgaagaa</td><td>ctgaacctgg</td><td>tgatcatcgg</td><td>cccgtctgct</td><td> 460</td>

<td>gatatcatcc</td><td>agttcgagtg</td><td>taagagcttt</td><td>ggtcacgaag</td><td>ttctgaacct</td><td>cacccgtaac</td><td> 540</td>

<td>ggctacggtt</td><td>ccactcagta</td><td>catccgtttc</td><td>tctccggact</td><td>tcaccttcgg</td><td>ttttgaagaa</td><td> 600</td>

<td>tccctggaag</td><td>tagacacgaa</td><td>cccactgctg</td><td>ggcgctggta</td><td>aattcgcaac</td><td>tgatcctgcg</td><td> 660</td>

<td>gttaccctgg</td><td>ctcacgaact</td><td>gattcatgca</td><td>ggccaccgcc</td><td>tgtacggtat</td><td>cgccatcaat</td><td> 720</td>

<td>ccgaaccgtg</td><td>tcttcaaagt</td><td>taacaccaac</td><td>gcgtattacg</td><td>agatgtccgg</td><td>tctggaagtt</td><td> 780</td>

<td>agcttcgaag</td><td>aactgcgtac</td><td>ttttggcggt</td><td>cacgacgcta</td><td>aattcatcga</td><td>ctctctgcaa</td><td> 840</td>

<td>gaaaacgagt</td><td>tccgtctgta</td><td>ctactataac</td><td>aagttcaaag</td><td>atatcgcatc</td><td>caccctgaac</td><td> 900</td>

102 aaagcgaaat gaaaaatacc gataaacttt aaagttctga gtgccgaaag gctaatttta Łtcactggtc ccatcgtggg tgctcagcga acaaaatgct accgcaaaac ttaactacac acggccagaa tgtccagctcgacta tactgccgga tgtccagctcgacta tactaccgga tga

Cctctccagt ggcaaattct tacaccgaag ttcgacaagg ggtttcaacc aacaacatga ctgtgcgtcg acatgaagaa ctgtagacaa acaacttcgt cagtattcaa tgcgtaacac acttcacaaa ac cgtttttaacaa taggacaa ta cgttttacaa cgtttttaacaa

960

1020

1080

1140

1200

1260

1302 &lt;210&gt; 2 &lt;211&gt; 1257 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 2

103 ctgcagtgta tcaaggttaa caactgggat Łtattcttca gcccgagtga agacaacttc € 0 accaacgacc tgaacaaagg tgaagaaatc acctcagata ctaacatcga agcagccgaa120 gaaaacatct cgctggacct gatccagcag tactacctga cctttaattt cgacaacgag180 ccggaaaaca tttctatcga aaacctgagc tctgatatca tcggccagct ggaactgatg240 ccgaacatcg aacgtttccc aaaccgtaaa aagtacgagc tggacaaata taccatgttc300 cactacctgc gcgcgcagga atttgaacac ggcaaatccc gtatcgcact gactaactcc360 gttaacgaag ctctgctcaa cccgtcccgt gtatacacct tcttctctag cgactacgtg420 aaaaaggtca acaaagcgac tgaagctgca atgttcttgg gttgggttga acagcttgtt480 tatgatttta ccgacgagac gtccgaagta tctactaccg acaaaattgc ggatatcact540 atcatcatcc cgtacatcgg tccggctctg aacattggca acatgctgta caaagacgac600 ttcgttggcg cactgatctt ctccggtgcg gtgatcctgc tggagttcat cccggaaatc660 gccatcccgg tactgggcac ctttgctctg gtttcttaca ttgcaaacaa ggttctgact720 gtacaaacca tcgacaacgc gctgagcaaa cgtaacgaaa aatgggatga agtttacaaa780 tatatcgtga ccaactggct ggctaaggtt aatactcaga tcgacctcat ccgcaaaaaa840 atgaaagaag cactggaaaa ccaggcggaa gctaccaagg caatcattaa ctacaagtac900 aaccagtaca ccgaggaaga aaaaaacaac atcaacttca acatcgacga tctgtcctct960 aaactgaacg aatccatcaa caaagctatg atcaacatca acaagttcct gaaccagtgc1020 tctgtaagct atctgatgaa ctccatgatc ccgtacggtg ttaaacgtct ggaggacttc1080 gatgcgtctc tgaaagacgc cctgctgaaa tacatttacg acaaccgtgg cactctgatc1140 ggtcaggttg atcgtctgaa ggacaaagtg aacaatacct tatcgaccga catccctttt1200 cagctcagta aatatgtcga taaccaacgc cttttgtcca ctctagacta gaagctt1257 &lt;210&gt; 3 &lt;211&gt; 1323 &lt;212 &gt; DNA &lt;213&gt; Dummy sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 3

104

<td>ggatccatgc</td><td>cggttaccat</td><td>caacaacttc</td><td>aactacaacg</td><td>acccgatcga</td><td>caacaacaac</td><td> 60</td>

<td>atcaCtatga</td><td>tggaaccgcc</td><td>gttcgcacgt</td><td>ggtaccggac</td><td>gttactacaa</td><td>ggcttttaag</td><td> 120</td>

<td>atcaccgacc</td><td>gtatctggat</td><td>catcccggaa</td><td>cgttacacct</td><td>tcggttacaa</td><td>acctgaggac</td><td>ISO</td>

<td>ttcaacaaga</td><td>gtagcgggat</td><td>tttcaaCcgt</td><td>gacgtctgcg</td><td>agtactatga</td><td>cccagattat</td><td> 240</td>

<td>ctgaatacca</td><td>acgataagaa</td><td>gaacatattc</td><td>cttcagacta</td><td>tgattaaact</td><td>cttcaaccgt</td><td> 300</td>

<td>atcaaaagca</td><td>aaccgctcgg</td><td>tgaaaaactc</td><td>ctcgaaatga</td><td>ttatcaacgg</td><td>tatcccgtac</td><td> 360</td>

<td>ctcggtgacc</td><td>gtcgtgtccc</td><td>gcttgaagag</td><td>ttcaacacca</td><td>acatcgcaag</td><td>cgtcaccgtc</td><td> 420</td>

<td>aacaaactca</td><td>tcagcaaccc</td><td>aggtgaagtc</td><td>gaacgtaaaa</td><td>aaggtatctt</td><td>cgcaaacctc</td><td> 400</td>

<td>atcatcttcg</td><td>gtccgggtcc</td><td>ggtcctcaac</td><td>gaaaacgaaa</td><td>ccatcgacat</td><td>cggtatccag</td><td> 540</td>

<td>aaccacttcg</td><td>caagccgtga</td><td>aggtttcggt</td><td>ggtatcatgc</td><td>agacgaaatt</td><td>ctgcccggaa</td><td> 600</td>

<td>tacgtcagtg</td><td>tcttcaacaa</td><td>cgtccaggaa</td><td>aacaaaggtg</td><td>caagcatctt</td><td>caaccgtcgt</td><td> 660</td>

<td>ggttacttca</td><td>gcgacccggc</td><td>actcatcctc</td><td>atgcatgaac</td><td>tcatccacgt</td><td>cctccacggt</td><td> 720</td>

<td>cCctacggta</td><td>tcaaagttga</td><td>cgacctcccg</td><td>atcgtcccga</td><td>acgagaagaa</td><td>attcttcatg</td><td> 700</td>

<td>cagagcaccg</td><td>acgcaatcca</td><td>ggctgaggaa</td><td>ctctacacct</td><td>tcggtggcca</td><td>agacccaagt</td><td> 840</td>

<td>atcataaccc</td><td>agtccaccga</td><td>caaaagcacc</td><td>tacgacaaag</td><td>Łcctccagaa</td><td>cttcaggggt</td><td> 900</td>

<td>atcgtggaca</td><td>gactcaacaa</td><td>agtcctcgtc</td><td>tgcatcagcg</td><td>acccgaacat</td><td>caatatcaac</td><td> 960</td>

<td>atatacaaga</td><td>acaagttcaa</td><td>agacaagtac</td><td>aaattcgtcg</td><td>aggacagcga</td><td>aggcaaatac</td><td> 1020</td>

<td>agcatcgacg</td><td>tagaaagtrtt</td><td>cgacaagctc</td><td>tacaaaagcc</td><td>tcatgttcgg</td><td>tttcaccgaa</td><td>1OS0</td>

<td>accaacatcg</td><td>ccgagaacta</td><td>caagatcaag</td><td>acaagggcaa</td><td>gttacttcag</td><td>cgacagcctc</td><td> 1140</td>

<td>ccgcctgtca</td><td>aaatcaagaa</td><td>cctcttagac</td><td>aacgagattt</td><td>acacaattga</td><td>agagggcttc</td><td> 1200</td>

<td>aacatcagtg</td><td>acaaagacat</td><td>ggagaaggaa</td><td>tacagaggtc</td><td>agaacaaggc</td><td>tatcaacaaa</td><td> 1260</td>

<td>caggcatacg</td><td>aggagatcag</td><td>caagaacac</td><td>ctcgcagtct</td><td>acaagatcca</td><td>gatgtgcgtc</td><td> 1320</td>

gac 1323 &lt;210&gt; 4 &lt;211&gt; 1260 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 4

105

<td>ctgcagtgca</td><td>tcgacgttga</td><td>caacgaagac ctgttcttca</td><td>tcgctgacaa</td><td>aaacagcttc</td><td>it's the</td>

<td>agtgacgacc</td><td>tgagcaaaaa</td><td>cgaacgtatc gaatacaaca</td><td>cccagagcaa</td><td>ctacatcgaa</td><td> 120</td>

<td>aacgacttcc</td><td>cgatcaacga</td><td>actgatcctg gacaccgacc</td><td>tgataagtaa</td><td>aaEcgaactg</td><td>iao</td>

<td>ccgagcgaaa</td><td>acaccgaaag</td><td>tctgaccgac ttcaacgttg</td><td>acgttccggt</td><td>ttacgaaaaa</td><td> 240</td>

<td>cagccggcta</td><td>tcaagaaaat</td><td>cttcaccgac gaaaacacca</td><td>tcttccagta</td><td>cctgtacagc</td><td> 300</td>

<td>cagaccttcc</td><td>cgctggacat</td><td>ccgtgacatc agtctgacca</td><td>gcagtttcga</td><td>cgacgctctg</td><td> 360</td>

<td>ctgttcagca</td><td>acaaagttta</td><td>cagtttcttc agcatggact</td><td>acatcaaaac</td><td>cgctaacaaa</td><td> 420</td>

<td>gttgttgaag</td><td>cagggctgtt</td><td>cgctggttgg gtEaaacaga</td><td>tcgttaacga</td><td>cttcgttatc</td><td> 480</td>

<td>gaagctaaca</td><td>aaagcaacac</td><td>tatggacaaa atcgctgaca</td><td>tcagtcEgat</td><td>cgttccgtac</td><td> 540</td>

<td>atcggtctgg</td><td>ctctgaacgt</td><td>tggtaacgaa accgctaaag</td><td>gtaacttcga</td><td>aaacgctttc</td><td> 600</td>

<td>gagatcgctg</td><td>gtgcaagcat</td><td>cctgctggag ttcatcccgg</td><td>aactgctgat</td><td>cccggttgtt</td><td> 660</td>

<td>ggtgctttcc</td><td>tgctggaaag</td><td>ttacatcgac aacaaaaaca</td><td>agatcatcaa</td><td>aaccatcgac</td><td> 720</td>

<td>aacgctctga</td><td>ccaaacgtaa</td><td>cgaaaaatgg agtgatatgt</td><td>acggtctgat</td><td>cgttgctcag</td><td> 780</td>

<td>tggctgagca</td><td>ccgtcaacac</td><td>ccagttctac accatcaaag</td><td>aaggtatgta</td><td>caaagctctg</td><td> 840</td>

<td>aactaccagg</td><td>ctcaggctct</td><td>ggaagagatc atcaaatacc</td><td>gttacaacat</td><td>ctacagtgag</td><td> 900</td>

<td>your area</td><td>gtaacatcaa</td><td>catcgacttc aacgacatca</td><td>acagcaaact</td><td>gaacgaaggt</td><td> 960</td>

<td>atcaaccagg</td><td>cEaEcgacaa</td><td>catcaacaac ttcatcaacg</td><td>gttgcagtgt</td><td>tagctacctg</td><td> 1020</td>

<td>atgaagaaga</td><td>tgatcccgct</td><td>ggctgttgaa aaactgctgg</td><td>acttcgacaa</td><td>caccctgaaa</td><td> 1080</td>

<td>aagaacctgc</td><td>tgaactacat</td><td>cgacgaaaac aagctgtacc</td><td>tgatcggtag</td><td>tgctgaatac</td><td> 1140</td>

<td>gaaaaaagta</td><td>aagtgaacaa</td><td>atacctgaag accatcatgc</td><td>cgttcgacct</td><td>gagtatctac</td><td> 1200</td>

<td>accaacgaca</td><td>ccatcctgat</td><td>cgaaatgttc aacaaataca</td><td>actctctaga</td><td>ctagaagctt</td><td> 1260</td>

&lt;210&gt; 5 &lt;211&gt; 1329 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 5

106 ggatccgaat tcatgccgat caccatcaac aacttcaact acagcgatcc ggtggataac aaaaacatcc Egtacctgga taeccatctg aataccctgg cgaacgaacc ggaaaaagcg EEEcgEatca ccggcaacat ttgggttatt ccggatcgtt ttagccgtaa cagcaacccg aatctgaata aaccgccgcg tgttaccagc ccgaaaagcg gttattacga tccgaactat ctgagcaccg atagcgataa agataccttc ctgaaagaaa tcatcaaact gttcaaacgc atcaacagcc gtgaaaEEgg cgaagaactg atcEatcgcc tgagcaccga tattccgttt ccgggcaaca acaacaCccc gatcaacacc tttgatttcg atgtggattt caacagcgtt gatgttaaaa cccgccaggg taacaattgg gtgaaaaccg gcagcattaa cccgagcgtg attattaccg gtccgcgcga aaacattatt gatccggaaa ccagcaccEt taaactgacc aacaacacct ttgcggcgca ggaaggtttt ggcgcgctga gcattattag cattagcccg cgctttatgc tgacctatag caacgcgacc aacgatgttg gtgaaggccg tttcagcaaa agcgaaEEEt gcatggaccc gatccEgatc ctgaŁgcatg aactgaacca tgcgatgcat aacctgtatg gcatcgcgat tccgaacgat cagaccatta gcagcgtgac cagcaacatc ttttacagcc agtacaacgt gaaactggaa tatgcggaaa tctatgcgtt tggcggtccg accattgatc tgattccgaa aagcgcgcgc aaatacttcg aagaaaaagc gctggattac tategcagea ttgcgaaacg tctgaacagc attaccaccg agaatccgag cagcttcaac aaatatatcg gcgaatataa acagaaactg atccgcaaat atcgctttgt ggtggaaagc agcggcgaag ttaccgttaa ccgcaataaa ttcgtggaac tgtacaacga actgacccag atcttcaccg aatttaacta tgcgaaaatc tataacgtgc agaaccgtaa aatctacctg agcaacgtgt ataccccggt gaccgcgaat attctggatg ataaagtgta cgatatccag aacggcttta acatcccgaa aagcaaectg aaegttctgt ttatgggcca gaacctgagc cgtaatccgg cgctgcgtaa agtgaacccg gaaaacatgc tgtacctgct caccaaattt tgcgtcgac

120

190

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1329 &lt;211&gt; 1263 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt;

&lt;400&gt;

Synthetic 6

107 ctgcagtgtc gtgaactgct ggtgaaaaac accgatctgc cgtttattgg cgatatcagc60 gatgtgaaaa ccgatatctt cctgcgcaaa gatatcaacg aagaaaccga agtgatctac 120 tacccggata acgtgagcgt tgatcaggtg atcctgagca aaaacaccag cgaacatggt190 cagctggatc tgctgtatcc gagcattgat agcgaaagcg aaattctgcc gggcgaaaac240 caggtgtttt acgataaccg tacccagaac gtggattacc tgaacagcta ttactacctg300 gaaagccaga aactgagcga taacgtggaa gattttacct ttacccgcag cattgaagaa360 gcgctggata acagcgcgaa agtttacacc tattttccga ccctggcgaa caaagttaat420 gcgggtgttc agggcggtct gtttctgatg tgggcgaacg atgtggtgga agatttcacc480 accaacatcc tgcgta aaga taccctggat aaaatcagcg atgttagcgc gattattccg540 tatattggtc cggcgctgaa cattagcaat agcgtgcgtc gtggcaattt taccgaagcg600 tttgcggtta ccggtgtgac cattctgctg gaagcgtttc cggaatttac cattccggcg660 ctgggtgcgt ttgtgatcta tagcaaagtg caggaącgca acgaaatcat caaaaccatc720 gataactgcc tggaacagcg tattaaacgc tgga of Agata gctatgaatg gatgatgggc780 acctggctga gccgtattat cacccagttc of the acatca gctaccagat gtacgatagcB40 ctgaactatc aggcgggtgc gattaaagcg aaaatcgatc tggaatacaa aaaatacagc900 ggcagcgata aagaaaacat caaaagccag gttgaaaacc tgaaaaacag cctggatgtg960 aaaattagcg aagcgatgaa taacatcaac aaattcatcc gcgaatgcag cgtgacctac1020 ctgttcaaaa acatgctgcc gaaagtgatc gatgaactga acgaatttga tcgcaacacc1000 aaagcgaaac tgatcaacct gatcgatage cacaacatta Ltctggtggg cgaagtggat1140 aaactgaaag cgaaagttaa caacagcttc cagaacacca tcccgtttaa catcttcagc1200 tataccaaca acagcctgct gaaagatatc atcaacgaat acttcaatct agactagaag1260

Ctt1263 &lt;210&gt; 7 &lt;211&gt; 207 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 7

108 ggatzccacgc acgtcgacgg catcattacc tccaaaacta aatctctgat cgaaggtcgt tttggcggtt tcacgggcgc acgcaaatca gcgcgtaaat tagctaacca ggcgctagcg 99cggtggcg gtagcggcgg tggcggtagc ggcggtggcg gtagcgcact agtgctgcag acgcacggtc tagaatgata aaagctt &lt;210&gt; 8 &lt;211&gt; 108 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 8 ggatccacgc acgtcgacgg catcattacc tccaaaacta aatctctgat agaaggtaga aacaaagcgc tgaacctgca gacgcacggt ctagaatgat aaaagctt &lt;210&gt; 9 &lt;211&gt; DNA &lt;211&gt; &lt;211&gt; &lt;211&gt; &lt;211&gt;

&lt;223&gt; Synthetic &lt;400&gt; 9 catatgaata acctcgggat tgagggtcgt tttggcggtt tcacgggcgc acgcaaatca gcgcgtaaat tagctaacca gactagtggc ggtgggggta gtggcggtttgg cgg999 99999^and gccctagggg atccgtcgac ctgcagggtc tagaagcgct agcgtgataa aagctt &lt;210&gt; 10 &lt;211&gt; 180 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 10 € 0

120

180

207

10S

120

180

186

109 ggatccacgc acgtcgacgc gattgatggt cgttttggcg gtttcacggg cgcacgcaaa tcagcgcgta aattagctaa ccaggcgcta gcgggcggtg gcggtagcgg cggtggcggt agcggcggtg gcggtagcgc aetagtgetg cagacgcacg gtctagaatg ataaaagctt &lt;210&gt; 11 &lt;211&gt; 249 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 11 ggatccacgc acgtcgacgg catcattacc tccaaaacta aatctctgat cgaaggtcgt tacggtggtt tcatgacctc tgaaaaatct cagaccccgc tggttacctc tgaaaaatct cagaccccgt tggttaccct gttcaacataaaacga gggtgtgtgtgactaaaa ga

120

ISO

120

180

240

249

<td>ggtggeggtt</td><td>ctggtggtgg</td><td>tggttctgca</td><td>CTagtgctgc</td><td>agacgcacgg</td><td>tctagaatga</td>

<td colspan="3">taaaagcct : 210&gt; 12 : 211&gt; 207 : 212&gt; DNA : 213&gt; Dummy sequence : 220&gt; : 223&gt; Synthetic : 400&gt; 12 ggatccacgc acgtcgacgg catcattacc</td><td>tccaaaacta</td><td>aatctctgat</td><td>cgaaggtcgt</td>

<td>tttggcggtt</td><td>tcacgggcgc</td><td>acgcaaatca</td><td>gcgcgtaaac</td><td>gtaagaacca</td><td>ggcgctagcg</td>

<td>ggcggtggcg</td><td>gtagcggcgg</td><td>tggcggtagc</td><td>ggcggtggcg</td><td>gtagcgcact</td><td>agtgctgcag</td>

<td>acgcacggtc</td><td>tagaatgata</td><td>aaagctt</td><td></td><td></td><td></td>

&lt;210&gt; 13 &lt;211&gt; 2709 &lt;212&gt; DNA

120

180

207 &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 13

110 ggatccatgg attgcttaca cacaacaaaa ctgaacccgc tctaccgata tactccaccg ggcggttcta cagccggacg gatatcatcc ggctacggtt tccctggaag gttaccctgg ccgaaccgtg agcttcgaag agttcgttaa caaacagttc aactataaag acccagttaa cggtgttgac tcaaaatccc gaacgctggc cagatgcagc cggtaaaggc attcaaaatc tctgggttat cccggaacgt gataccttta ctaacccgga agaaggtgac eaccggaagc gaaacaggtg ccggtatctt actatgactc cacctacctg acgaaaagga caactacctg aaaggtgtta ctaaactgtt cgagcgtatt acctgggccg tatgctgctg actagcatcg ttcgcggtat cccgttctgg ccatcgatac cgaactgaaa gtaatcgaca ctaactgcat caacgCtatt gttcctatcg ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct agttcgagtg taagagcttt ggtcacgaag ttctgaacct cacccgtaac ccactcagta catccgtttc tctccggact tcaccttcgg ttttgaagaa tagacacgaa cccactgctg ggcgcCggta aattcgcaac tgatcctgcg ctcacgaact gattcatgca ggccaccgcc tgtacggtat cgccatcaat tcttcaaagt Caacaccaac gcgtattacg agatgtccgg tctggaagtt aactgcgtac ttttggcggt cacgacgcta aattcatcga ctctctgcaa

120

180

240

300

360

420

4B0

540

600

660

720

7B0

840

111

<td>gaaaacgagt</td><td>tccgtctgta</td><td>ctactataac</td><td>aagttcaaag</td><td>atatcgcatc</td><td>caccctgaac</td><td> 900</td>

<td>aaagcgaaat</td><td>ccatcgtggg</td><td>taccactgct</td><td>tctctccagt</td><td>acatgaagaa</td><td>cgtttttaaa</td><td> 960</td>

<td>gaaaaatacc</td><td>tgctcagcga</td><td>agacacctcc</td><td>ggcaaattct</td><td>ctgtagacaa</td><td>gttgaaattc</td><td> 1020</td>

<td>gataaacttt</td><td>acaaaatgct</td><td>gactgaaatt</td><td>tacaccgaag</td><td>acaacttcgt</td><td>taagttcttt</td><td> 1000</td>

<td>aaagttctga</td><td>accgcaaaac</td><td>ctatctgaac</td><td>ttcgacaagg</td><td>cagtattcaa</td><td>aatcaacatc</td><td> 1140</td>

<td>gtgccgaaag</td><td>ttaactacac</td><td>tatctacgat</td><td>ggtttcaacc</td><td>tgcgtaacac</td><td>aaacctggct</td><td> 1200</td>

<td>gctaatttta</td><td>acggccagaa</td><td>cacggaaatc</td><td>aacaacatga</td><td>acttcacaaa</td><td>actgaaaaac</td><td> 1260</td>

<td>ttcactggtc</td><td>tgttcgagtt</td><td>ttacaagctg</td><td>ctgtgcgtcg</td><td>acggcatcat</td><td>tacctccaaa</td><td> 1320</td>

<td>actaateCtC</td><td>tgatagaagg</td><td>tagatttggc</td><td>ggtttcacgg</td><td>gcgcacgcaa</td><td>atcagcgcgt</td><td> 1380</td>

<td>aaattagcta</td><td>accaggcgct</td><td>Gcgggcggt</td><td>ggcggtagcg</td><td>gcggtggcgg</td><td>tagcggcggt</td><td> 1440</td>

<td>ggcggtagcg</td><td>caccagcgcc</td><td>gcagcgtatc</td><td>aaggttaaca</td><td>actgggattt</td><td>attcttcagc</td><td> 1500</td>

<td>ccgagtgaag</td><td>acaacttcac</td><td>caacgacctg</td><td>aacaaaggtg</td><td>aagaaatcac</td><td>ctcagatact</td><td> 1560</td>

<td>aacatcgaag</td><td>your foot</td><td>aaacatctcg</td><td>ctggacctga</td><td>tccagcagta</td><td>ctacctgacc</td><td> 1620</td>

<td>tttaatttcg</td><td>acaacgagcc</td><td>ggaaaacatt</td><td>tctatcgaaa</td><td>acctgagctc</td><td>tgatatcatc</td><td> 1680</td>

<td>ggccagctgg</td><td>aactgatgcc</td><td>gaacatcgaa</td><td>cgtttcccaa</td><td>acggtaaaaa</td><td>gtacgagctg</td><td> 1740</td>

<td>hand</td><td>ccatgttcca</td><td>ctacctgcgc</td><td>gcgcaggaat</td><td>ttgaacacgg</td><td>caaatcccgt</td><td> 1800</td>

<td>atcgcactga</td><td>ctaactccgt</td><td>taacgaagct</td><td>ctgctcaacc</td><td>cgtcccgtgt</td><td>atacaccttc</td><td> 1860</td>

<td>ttctctagcg</td><td>actacgtgaa</td><td>aaaggtcaaa</td><td>aaagcgactg</td><td>aagctgcaat</td><td>gttcttgggt</td><td> 1920</td>

<td>tgggttgaac</td><td>agcttgttta</td><td>tgattttacc</td><td>gacgagacgt</td><td>ccgaagtatc</td><td>tactaccgac</td><td> 1980</td>

<td>aaaattgcgg</td><td>so cactus</td><td>catcatcccg</td><td>tacatcggtc</td><td>cggctctgaa</td><td>cattggcaac</td><td> 2040</td>

<td>atgccgtaca</td><td>aagacgactt</td><td>cgttggcgca</td><td>ctgatcttct</td><td>ccggtgcggt</td><td>gatcctgctg</td><td> 2100</td>

<td>gagttcatcc</td><td>cggaaatcgc</td><td>catcccggta</td><td>ctgggcacct</td><td>ttgctctggt</td><td>ttcttacatt</td><td> 2160</td>

<td>gcaaacaagg</td><td>ttctgactgt</td><td>acaaaccatc</td><td>gacaacgcgc</td><td>tgagcaaacg</td><td>taacgaaaaa</td><td> 2220</td>

<td>tgggatgaag</td><td>tttacaaata</td><td>tatcgtgacc</td><td>aactggctgg</td><td>ctaaggttaa</td><td>tactaagatc</td><td> 2280</td>

<td>gacctcatcc</td><td>gcaaaaaaat</td><td>the heat</td><td>ctggaaaacc</td><td>aggcggaagc</td><td>taccaaggca</td><td> 2340</td>

<td>atcattaact</td><td>accagtacaa</td><td>ccagtacacc</td><td>hurry</td><td>aaaacaacat</td><td>caacttcaac</td><td> 2400</td>

<td>atcgacgatc</td><td>tgtcctaa</td><td>actgaacgaa</td><td>tccatcaaca</td><td>aagctatgat</td><td>caacatcaac</td><td> 2460</td>

<td>aagttcctga</td><td>accagtgctc</td><td>tgtaagctat</td><td>ctgatgaact</td><td>ccatgatccc</td><td>gtacggtgtt</td><td> 2520</td>

<td>aaacgtctgg</td><td>aggacttcga</td><td>tgcgtctctg</td><td>aaagacgccc</td><td>tgctgaaata</td><td>catttacgac</td><td> 2580</td>

<td>aaccgtggca</td><td>ctctgatcgg</td><td>tcaggttgat</td><td>cgtctgaagg</td><td>acaaagtgaa</td><td>caatacctta</td><td> 2640</td>

<td>tcgaccgaca</td><td>tcccttttca</td><td>gctcagtaaa</td><td>tatgtcgata</td><td>accaacgcct</td><td>tttgtccact</td><td> 2700</td>

112 ctagactag &lt;210&gt; 14 &lt;211&gt; 902 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 14

113

Gly Ser Met Glu Phe Val Asn Lys Gin Phe Asn Tyr Łys Asp Pro Val

5 1015

Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gin Met 20 2S30

Gin Pro val Lys Ala Phe Lys Ile His Asn Ly s Ile Trp val Ile pro 35 4045

Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro 50 5560

Pro Glu Ala Lys Gin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu 65 70 7580

Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu 85 9095

Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser 100 105 110

Ile Val Arg Gly ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu 115 120 125

Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gin Pro Asp Gly

130 135140

Tyr Arg Cheese Glu Glu Leu Asn Leu Val Ile Gly Pro Ser Ala 145 150 155160

Asp Ile Ile Gin Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn

165 170175

Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gin Tyr Ile Arg Phe Ser Pro 180 18S190

114

<td>Asp</td><td>Phe</td><td colspan="2">Thr Phe 195</td><td>Gly</td><td>Phe</td><td>Glu</td><td>Glu 200</td>

<td>Leu</td><td>Leu 210</td><td>Gly</td><td>Alu</td><td>Gly</td><td>List</td><td>Phe 215</td><td>Ala</td>

<td>His 225</td><td>Glu</td><td>Leu</td><td>How much</td><td>His</td><td>Ala 230</td><td>Gly</td><td>His</td>

<td>Pro</td><td>Asn</td><td>Arg</td><td>Val</td><td>Phe 245</td><td>List</td><td>Val</td><td>Asn</td>

<td>Gly</td><td>Leu</td><td>Glu</td><td>Val 260</td><td>Cheese</td><td>Phe</td><td>Glu</td><td>Glu</td>

<td>Al a</td><td>List</td><td>Phe 275</td><td>How much</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Gin 280</td>

<td>Tyr</td><td>Asn 290</td><td>List</td><td>Phe</td><td>List</td><td>Asp</td><td>How much 295</td><td>Ala</td>

<td>How much 305</td><td>Val</td><td>Gly</td><td>Thr</td><td>Thr</td><td>Ala 310</td><td>Cheese</td><td>Leu</td>

<td>G1U</td><td>List</td><td>Tyr</td><td>Leu</td><td>Leu 325</td><td>Cheese</td><td>Glu</td><td>Asp</td>

<td>List</td><td>Leu</td><td>List</td><td>Phe 340</td><td>Asp</td><td>List</td><td>Leu</td><td>Tyr</td>

<td>Glu</td><td>Asp</td><td>Asn 355</td><td>Phe</td><td>Val</td><td>List</td><td>Phe</td><td>Phe 360</td>

<td>Leu</td><td>Asn 370</td><td>Phe</td><td>Asp</td><td>List</td><td>Ala</td><td>or 375</td><td>Phe</td>

<td>Asn 385</td><td>Tyr</td><td>Thr</td><td>How much</td><td>Tyr</td><td>Asp 390</td><td>Gly</td><td>Phe</td>

<td>Ala</td><td>Asn</td><td>Phe</td><td>Asn</td><td>Gly 405</td><td>Gin</td><td>Asn</td><td>Thr</td>

<td>List</td><td>Leu</td><td>List</td><td>Asn 420</td><td>Phe</td><td>Thr</td><td>Gly</td><td>Leu</td>

<td>Leu</td><td>Glu</td><td>Val</td><td>Asp 205</td><td>Thr</td><td>Asn</td><td>Pro</td>

<td>Asp</td><td>Pro</td><td>Ala 220</td><td>Val</td><td>Thr</td><td>Leu</td><td>Ala</td>

<td>Leu</td><td>Tire 235</td><td>Gly</td><td>How much</td><td>Ala</td><td>How much</td><td>Asn 240</td>

<td>Asn 250</td><td>Ala</td><td>Tyr</td><td>Tyr</td><td>Glu</td><td>Met 255</td><td>Cheese</td>

<td>Arg</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Gly 270</td><td>His</td><td>Asp</td>

<td>Asn</td><td>Glu</td><td>Phe</td><td>Arg 285</td><td>Leu</td><td>Tyr</td><td>Tyr</td>

<td>Thr</td><td>Leu</td><td>Asn 300</td><td>List</td><td>Ala</td><td>List</td><td>Cheese</td>

<td>Tyr</td><td>Underworld 315</td><td>List</td><td>Asn</td><td>Val</td><td>Phe</td><td>Lys 320</td>

<td>Cheese 330</td><td>Gly</td><td>List</td><td>Phe</td><td>Cheese</td><td>Val 335</td><td>Asp</td>

<td>Underworld</td><td>Leu</td><td>Thr</td><td>G1U</td><td>How much 350</td><td>Tyr</td><td>Thr</td>

<td>Val</td><td>Leu</td><td>Asn</td><td>Arg 365</td><td>List</td><td>Thr</td><td>Tyr</td>

<td>How much</td><td>Asn</td><td>How much 380</td><td>Val</td><td>Pro</td><td>List</td><td>Val</td>

<td>Leu</td><td>Arg 395</td><td>Asn</td><td>Thr</td><td>Asn</td><td>Leu</td><td>Ala 400</td>

<td>How much 410</td><td>Asn</td><td>Asn</td><td>Underworld</td><td>Asn</td><td>Phe 415</td><td>Thr</td>

<td>Glu</td><td>Phe</td><td>Tyr</td><td>List</td><td>Leu 430</td><td>Leu</td><td>Cys</td>

115

<td>Val</td><td>Asp</td><td>Gly 435</td><td>How much</td><td>How much</td><td>Thr</td><td>Cheese</td><td>List 440</td><td>Thr</td><td>List</td><td>Cheese</td><td>Leu</td><td>How much 445</td><td>Glu</td><td>Gly</td><td>Arg</td>

<td>Phe</td><td>Gly 4 50</td><td>Gly</td><td>Phe</td><td>Thr</td><td>Gly</td><td>Ala 455</td><td>Arg</td><td>Bald</td><td>Cheese</td><td>Ala</td><td>Arg 460</td><td>List</td><td>Leu</td><td>Ala</td><td>Asn</td>

<td>Gin 455</td><td>Ala</td><td>Leu</td><td>Ala</td><td>Gly</td><td>Gly 470</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Gly 475</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Gly 480</td>

<td>Gly</td><td>Gly</td><td>Cheese</td><td>Ala</td><td>Leu 435</td><td>Val</td><td>Leu</td><td>Gin</td><td>Cys</td><td>He 490</td><td>List</td><td>Val</td><td>Asn</td><td>Asn</td><td>Trp 495</td><td>Asp</td>

<td>Leu</td><td>Phe</td><td>Phe</td><td>Cheese 500</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>Asp</td><td>Asn 505</td><td>Phe</td><td>Thr</td><td>Asn</td><td>Asp</td><td>Leu 510</td><td>Asn</td><td>List</td>

<td>Gly</td><td>Glu</td><td>Glu 515</td><td>How much</td><td>Thr</td><td>Cheese</td><td>Asp</td><td>Thr 520</td><td>Asn</td><td>How much</td><td>Glu</td><td>Ala</td><td>Ala 525</td><td>Glu</td><td>Glu</td><td>Asn</td>

<td>How much</td><td>Cheese 330</td><td>Leu</td><td>Asp</td><td>Leu</td><td>How much</td><td>Gin 535</td><td>Gin</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>Thr 540</td><td>Phe</td><td>Asn</td><td>Phe</td><td>ASp</td>

<td>Asn 545</td><td>G1U</td><td>Pro</td><td>Glu</td><td>Asn</td><td>How much 55 0</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Asn</td><td>Leu 555</td><td>Cheese</td><td>Cheese</td><td>Asp</td><td>How much</td><td>How much 560</td>

<td>Gly</td><td>Gin</td><td>Leu</td><td>Glu</td><td>Leu 565</td><td>Underworld</td><td>Pro</td><td>Asn</td><td>How much</td><td>Glu 570</td><td>Arg</td><td>Phe</td><td>Pro</td><td>Asn</td><td>Gly 575</td><td>List</td>

<td>List</td><td>Tyr</td><td>Glu</td><td>Leu Sao</td><td>Asp</td><td>List</td><td>Tyr</td><td>Thr</td><td>Underworld 535</td><td>Phe</td><td>His</td><td>Tyr</td><td>Leu</td><td>Arg 590</td><td>Ala</td><td>Gin</td>

<td>Glu</td><td>Phe</td><td>Glu S95</td><td>His</td><td>Gly</td><td>List</td><td>Cheese</td><td>Arg 600</td><td>How much</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Asn 605</td><td>Cheese</td><td>Val</td><td>Asn</td>

<td>Glu</td><td>Ala 610</td><td>Leu</td><td>Leu</td><td>Asn</td><td>Pro</td><td>Cheese 615</td><td>Arg</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Phe 620</td><td>Phe</td><td>Cheese</td><td>Cheese</td><td>Asp</td>

<td>Tire 625</td><td>Val</td><td>List</td><td>List</td><td>Val</td><td>Asn 630</td><td>List</td><td>Ala</td><td>Thr</td><td>Glu</td><td>Ala 635</td><td>Ala</td><td>Underworld</td><td>Phe</td><td>Leu</td><td>Gly 64 0</td>

<td>Trp</td><td>Val</td><td>Glu</td><td>Gin</td><td>Leu 645</td><td>Val</td><td>Tyr</td><td>ASp</td><td>Phe</td><td>Thr 650</td><td>Asp</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>Glu 655</td><td>Val</td>

<td>Cheese</td><td>Thr</td><td>Thr</td><td>Asp 660</td><td>List</td><td>He</td><td>Ala</td><td>Asp</td><td>How much 665</td><td>Thr</td><td>How much</td><td>Background</td><td>How much</td><td>Pro, 670</td><td>Tyr</td><td>How much</td>

<td>Gly</td><td>Pro</td><td>Ala</td><td>Leu</td><td>Asn</td><td>Background</td><td>Gly</td><td>Asn</td><td>Underworld</td><td>Leu</td><td>Tyr</td><td>List</td><td>ASp</td><td>Asp</td><td>Phe</td><td>Val</td>

116

675

680

685

<td>Gly</td><td>Ala 690</td><td>Leu</td><td>how much</td><td>Phe</td><td>Cheese</td><td>Gly 695</td><td>Ala</td><td>val</td><td>How much</td><td>Leu</td><td>Leu 700</td><td>Glu</td><td>Phe</td><td>How much</td>

<td>Glu 705</td><td>Background</td><td>Ala</td><td>How much</td><td>Pro</td><td>Val 710</td><td>Leu</td><td>Gly</td><td>Thr</td><td>Phe</td><td>Ala 715</td><td>Leu</td><td>Val</td><td>Cheese</td><td>Tyr</td>

<td>Ala</td><td>Asn</td><td>List</td><td>Val</td><td>Leu 725</td><td>Thr</td><td>Val</td><td>Gin</td><td>Thr</td><td>How much 730</td><td>Asp</td><td>Asn</td><td>Ala</td><td>Leu</td><td>cheese 735</td>

<td>Arg</td><td>Asn</td><td>Glu</td><td>List 740</td><td>Trp</td><td>Asp</td><td>Glu</td><td>Val</td><td>Tire 745</td><td>List</td><td>Tyr</td><td>Background</td><td>Val</td><td>Thr 750</td><td>Asn</td>

<td>Leu</td><td>Ala</td><td>Lys 755</td><td>val</td><td>Asn</td><td>Thr</td><td>Gin</td><td>How much 760</td><td>Asp</td><td>Leu</td><td>How much</td><td>Arg</td><td>Lys 765</td><td>List</td><td>Underworld</td>

<td>GLU</td><td>Ala 770</td><td>Leu</td><td>Glu</td><td>Asn</td><td>Gin</td><td>Ala 775</td><td>Glu</td><td>Ala</td><td>Thr</td><td>List</td><td>Ala 780</td><td>How much</td><td>How much</td><td>Asn</td>

<td>Gin 7B5</td><td>Tyr</td><td>Asn</td><td>Gin</td><td>Tyr</td><td>Thr 790</td><td>Glu</td><td>Glu</td><td>Glu</td><td>List</td><td>Asn 795</td><td>Asn</td><td>How much</td><td>Asn</td><td>Phe</td>

<td>How much</td><td>Asp</td><td>Asp</td><td>Leu</td><td>Cheese 805</td><td>Cheese</td><td>List</td><td>Leu</td><td>Asn</td><td>Glu Θ10</td><td>Cheese</td><td>How much</td><td>Asn</td><td>List</td><td>Area B15</td>

<td>how much</td><td>Asn</td><td>How much</td><td>Asn 820</td><td>List</td><td>Phe</td><td>Leu</td><td>Asn</td><td>Gin 825</td><td>Cys</td><td>Cheese</td><td>Val</td><td>Cheese</td><td>Tire 830</td><td>Leu</td>

<td>Asn</td><td>Cheese</td><td>Met 835</td><td>How much</td><td>Pro</td><td>Tyr</td><td>Gly</td><td>Val 840</td><td>List</td><td>Arg</td><td>Leu</td><td>Glu</td><td>Asp 845</td><td>Phe</td><td>Asp</td>

<td>Cheese</td><td>Leu 850</td><td>List</td><td>Asp</td><td>Ala</td><td>Leu</td><td>Leu 855</td><td>List</td><td>Tyr</td><td>How much</td><td>Tyr</td><td>Asp B60</td><td>Asn</td><td>Arg</td><td>Gly</td>

<td>Leu 8 65</td><td>How much</td><td>Gly</td><td>Gin</td><td>Val</td><td>Asp 870</td><td>Arg</td><td>Leu</td><td>List</td><td>Asp</td><td>Lys 875</td><td>Val</td><td>Asn</td><td>Asn</td><td>Thr</td>

<td>Cheese</td><td>Thr</td><td>Asp</td><td>How much</td><td>Pro 885</td><td>Phe</td><td>Gin</td><td>Leu</td><td>Cheese</td><td>Lys Θ90</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Asn</td><td>Gin 895</td>

Pro

How much 720

List

Trp

List

Tyr

Asn

800

Underworld

Underworld

Ala

Thr

Leu 880

Arg

Leu Leu Ser Thr Leu Asp

900

210&gt; 15 &lt;211&gt; 2736

117 &lt;212&gt;

&lt;213&gt;

&lt;220&gt;

&lt;223&gt;

&lt;400&gt;

GOUT

Artificial sequence

Synthetic

118

<td>ctcgggattg</td><td>agggtcgttt</td><td>tggcggtttc</td><td>acgggcgcac</td><td>gcaaatcagc</td><td>gcgtaaatta</td><td> 60</td>

<td>gctaaccaga</td><td>ctagtggcgg</td><td>tgggggtagt</td><td>ggcggtggcg</td><td>gttcgggcgg</td><td>gggtgggagc</td><td> 120</td>

<td>cctaggggat</td><td>ccatggagtt</td><td>cgttaacaaa</td><td>cagttcaact</td><td>ataaagaccc</td><td>agttaacggt</td><td> 130</td>

<td>gttgacattg</td><td>cttacatcaa</td><td>aatcccgaac</td><td>gctggccaga</td><td>tgcagccggt</td><td>aaaggcattc</td><td> 240</td>

<td>aaaatccaca</td><td>acaaaatctg</td><td>ggttatcccg</td><td>gaacgtgata</td><td>cctttactaa</td><td>cccggaagaa</td><td> 300</td>

<td>ggtgacctga</td><td>acccgccacc</td><td>ggaagcgaaa</td><td>caggtgccgg</td><td>tatcttacta</td><td>tgactccacc</td><td> 360</td>

<td>tacctgtcta</td><td>ccgataacga</td><td>aaaggacaac</td><td>tacctgaaag</td><td>gtgttactaa</td><td>actgttcgag</td><td> 420</td>

<td>cgtatttact</td><td>ccaccgacct</td><td>gggccgtatg</td><td>ctgctgacta</td><td>gcatcgttcg</td><td>cggtatcccg</td><td> 480</td>

<td>ttctggggcg</td><td>gttctaccat</td><td>cgataccgaa</td><td>ctgaaagtaa</td><td>tcgacactaa</td><td>ctgcatcaac</td><td> 540</td>

<td>gttattcagc</td><td>cggacggttc</td><td>ctatcgttcc</td><td>gaagaactga</td><td>acctggtgat</td><td>catcggcccg</td><td> 600</td>

<td>tctgctgata</td><td>tcatccagtt</td><td>cgagtgtaag</td><td>agctttggtc</td><td>acgaagttct</td><td>gaacctcacc</td><td> 660</td>

<td>cgtaacggct</td><td>acggttccac</td><td>tcagtacatc</td><td>cgtttctctc</td><td>cggacttcac</td><td>cttcggtttt</td><td> 720</td>

<td>gaagaatccc</td><td>tggaagtaga</td><td>cacgaaccca</td><td>ctgctgggcg</td><td>ctggtaaatt</td><td>cgcaactgat</td><td> 780</td>

<td>cctgcggtta</td><td>ccctggctca</td><td>cgaactgatt</td><td>catgcaggcc</td><td>accgcctgta</td><td>cggtatcgcc</td><td> 840</td>

<td>atcaatccga</td><td>accgtgtctt</td><td>caaagttaac</td><td>accaacgcgt</td><td>attacgagat</td><td>gtccggtctg</td><td> 900</td>

<td>gaagttagct</td><td>tcgaagaact</td><td>gcgtactttt</td><td>ggcggtcacg</td><td>acgctaaatt</td><td>catcgactct</td><td> 960</td>

<td>ctgcaagaaa</td><td>acgagttccg</td><td>tctgtactac</td><td>tataacaagt</td><td>tcaaagatat</td><td>cgcatccacc</td><td> 1020</td>

<td>ctgaacaaag</td><td>cgaaatccat</td><td>cgtgggtacc</td><td>actgcttctc</td><td>tccagtacat</td><td>gaagaacgtt</td><td> 1080</td>

<td>tttaaagaaa</td><td>aatacctgct</td><td>cagcgaagac</td><td>acctccggca</td><td>aattctctgt</td><td>agacaagttg</td><td> 1140</td>

<td>aaattcgata</td><td>aactttacaa</td><td>aatgctgact</td><td>gaaatttaca</td><td>ccgaagacaa</td><td>cttcgttaag</td><td> 1200</td>

<td>ttctttaaag</td><td>ttctgaaccg</td><td>caaaacctat</td><td>ctgaacttcg</td><td>acaaggcagt</td><td>attcaaaatc</td><td> 1260</td>

<td>aacatcgtgc</td><td>cgaaagttaa</td><td>ctacactatc</td><td>tacgatggtt</td><td>tcaacctgcg</td><td>taacaccaac</td><td> 1320</td>

<td>ctggctgcta</td><td>attttaacgg</td><td>ccagaacacg</td><td>gaatcaaca</td><td>acatgaactt</td><td>cacaaaactg</td><td> 1380</td>

<td>aaaaacttca</td><td>ctggtctgtt</td><td>cgagttttac</td><td>aagctgctgt</td><td>gcgtcgacgg</td><td>catcattacc</td><td> 1440</td>

<td>tccaaaacta</td><td>aatctctgat</td><td>agaaggtaga</td><td>aacaaagcgc</td><td>tgaacgacct</td><td>ctgtatcaag</td><td> 1500</td>

<td>gttaacaact</td><td>gggatttatt</td><td>cttcagcccg</td><td>agtgaagaca</td><td>acttcaccaa</td><td>cgacctgaac</td><td> 1560</td>

aaaggtgaag aaatcacctc agatactaac atcgaagcag ccgaagaaaa catctcgctg 1620

119

<td>gacctgatcc</td><td>agcagtacta</td><td>cctgaccttt</td><td>aatttcgaca</td><td>acgagccgga</td><td>aaacatttct</td><td> 1680</td>

<td>atcgaaaacc</td><td>tgagctctga</td><td>tatcatcggc</td><td>cagetggaac</td><td>tgatgccgaa</td><td>catcgaaegC</td><td> 1740</td>

<td>ttcccaaacg</td><td>gtaaaaagta</td><td>cgagctggac</td><td>aaatatacca</td><td>tgttccacta</td><td>cctgcgcgcg</td><td> 1800</td>

<td>caggatttg</td><td>aacacggcaa</td><td>atcccgtatc</td><td>gcactgacta</td><td>actccgttaa</td><td>cgaagctctg</td><td> 1660</td>

<td>ctcaacccgt</td><td>cccgtgtata</td><td>caccttcttc</td><td>tctagcgact</td><td>acgtgaaaaa</td><td>ggtcaacaaa</td><td> 1920</td>

<td>gcgactgaag</td><td>ctgcaatgtt</td><td>cttgggttgg</td><td>gttgaacagc</td><td>ttgtttatga</td><td>ttttacegac</td><td> 1980</td>

<td>gagacgtccg</td><td>aagtatctac</td><td>taccgacaaa</td><td>attgcggata</td><td>tcactatcat</td><td>catcccgtac</td><td> 2040</td>

<td>atcggtccgg</td><td>ctctgaacat</td><td>tggcaacatg</td><td>ctgtaca layer</td><td>acgacttcgt</td><td>tggcgcactg</td><td> 2100</td>

<td>atcttctccg</td><td>gtgcggtgat</td><td>cctgctggag</td><td>ttcatcccgg</td><td>aaatcgccat</td><td>cccggtactg</td><td> 2160</td>

<td>ggcacctttg</td><td>ctctggtttc</td><td>ttacattgca</td><td>aacaaggttc</td><td>tgactgtaca</td><td>aaccatcgac</td><td> 2220</td>

<td>aacgcgctga</td><td>gcaaacgtaa</td><td>cgaaaaatgg</td><td>gatgaagEtt</td><td>acaaatatat</td><td>cgtgaccaac</td><td> 2280</td>

<td>tggctggcta</td><td>aggttaatac</td><td>tcagatcgac</td><td>ctcatccgca</td><td>aaaaaatgaa</td><td>agaagcactg</td><td> 2340</td>

<td>gaaaaccagg</td><td>cggaagctac</td><td>caaggcaatc</td><td>attaactacc</td><td>agtacaacca</td><td>gtacaccgag</td><td> 2400</td>

<td>gaagaaaaaa</td><td>acaacatcaa</td><td>cttcaacatc</td><td>gacgatctgt</td><td>cctctaaact</td><td>gaacgatcc</td><td> 2460</td>

<td>atcaaeaaag</td><td>ctatgatcaa</td><td>catcaacaag</td><td>ttcctgaacc</td><td>agtgctctgt</td><td>aagctatctg</td><td> 2520</td>

<td>atgaactcca</td><td>tgatcccgta</td><td>aggtgttaaa</td><td>cgtctggagg</td><td>acttcgatgc</td><td>gtctctgaaa</td><td> 2580</td>

<td>gacgccctgc</td><td>tgaaatacat</td><td>ttacgacaac</td><td>cgtggcactc</td><td>tgatcggtca</td><td>ggttgatcgt</td><td> 2640</td>

<td>ctgaaggaca</td><td>aagtgaacaa</td><td>taccttatcg</td><td>accgacatcc</td><td>cttttcagct</td><td>cagtaaatat</td><td> 2700</td>

<td>gtcgataacc</td><td>aacgcctttt</td><td>gtccactcta</td><td>gactag</td><td></td><td></td><td> 2736</td>

&lt;210&gt; 16 &lt;211&gt; 911 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt;

Synthetic &lt;400&gt; 16

120

<td rowspan="2">Leu 1</td><td colspan="2" rowspan="2">Gly How many</td><td colspan="2" rowspan="2">Glu Gly 5</td><td rowspan="2">Arg</td><td rowspan="2">Phe</td><td colspan="8">Gly Gly Phe Thr Gly Ala Arg Lys Ser</td>

<td colspan="4"> 10</td><td colspan="4"> 15</td>

<td>Ala</td><td>Arg</td><td>List</td><td>Leu 20</td><td>Ala</td><td>Asn</td><td>Gin</td><td>Thr</td><td>Cheese 25</td><td>Gly Gly</td><td>Gly</td><td>Gly</td><td>Cheese 30</td><td>Gly</td><td>Gly</td>

<td>Gly</td><td>Gly</td><td>Cheese 35</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Cheese 40</td><td>Pro</td><td>Arg Gly</td><td>Cheese</td><td>Met 45</td><td>Glu</td><td>Phe</td><td>Val</td>

121

Asn Lys 50

Tyr Ile 65

Lys Ile

Gin Phe Asn Tyr Lys Asp Pro Val 55

Lys Ile Pro Asn Ala Gly Gin Met 70

His Asn Lys Ile Trp Val Ile Pro. ... 85. ..... 90

Asn Gly Val Asp Ile Ala 60

Gin Pro Val Lys Ala Phe 75 80

Glu Arg Asp Thr Phe Thr 95

Asn Pro Glu Glu Gly Asp Leu Asn 100

Pro Pro Pro Glu Ala Lys Gin Val 105 110

Pro Val Ser Tyr Tyr Asp Ser Thr

115 120

Tyr Leu Ser Thr Asp Asn Glu Lys 12S

Asp Asn Tyr Leu Lys Gly Val Thr

130 135

Lys Leu Phe Glu Arg Ile Tyr Ser

140

Thr Asp Leu Gly Arg Met Leu Leu 145 150

Thr Ser ile Val Arg Gly Ile Pro

155 160

Phe Trp Gly Gly Ser Thr Ile Asp

165

Thr Glu Leu Lys Val Ile Asp Thr

170 175

Asn Cys Ile Asn Val With Gin. Pro ISO

Asp Gly Cheese Tyr Arg Ser Glu Glu 185 190

Leu Asn Leu Val Ile Ile Gly Pro

195 200

Ala Asp ile Ile Gin Phe Glu cheese

205

Cys Lys Ser Phe Gly His Glu Val

210 215

Leu Asn Leu Thr Arg Asn Gly Tyr

220

Gly Ser Thr Gin Tyr Ile Arg Phe 225 230

Pro Asp Phe Thr Phe Gly Phe cheese

235 240

Glu Glu Ser Leu Glu Val Asp Thr

245

Asn Pro Leu Leu Gly Ala Gly Lys 250 255

Phe Ala Thr Asp Pro Ala Val Thr

260

Leu Ala His Glu Leu Ile His Ala 265 270

Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn Arg Val Phe Lys

275 260 285

Val Asn Thr Asn. Ala Tyr Tyr Glu Met Ser Gly Leu Glu Val Ser Phe

122

Asp Ala Lys Phe Ile Asp Ser

315 320

290 295

300

Glu Glu Leu Arg Thr Phe Gly Gly 305 310

Leu Gin Glu Asn Glu Phe Arg Leu 325

Tyr Tyr Asn Lys Phe Lys Asp 330 335

Ile Ala Ser Thr Leu Asn Lys Ala 340

Cheese Ile Val Gly Thr Thr Ala

350

Leu Gin Tyr Met Lys Asn Val cheese

355 360

Lys Glu Lys Tyr Leu Leu Ser 365

Glu Asp Thr Ser Gly Lys Phe Ser

370 375

Asp Lys Leu Lys Phe Asp Lys

380

Leu Tyr Lys Met Leu Thr Glu Ile 385 390

Thr Glu Asp Asn Phe Val Lys

395 400

Phe Phe Lys Val Leu Asn Arg Lys 405

Tyr Leu Asn Phe Asp Lys Ala 410 415

Asn ile Val Pro With Val Phe Lys

420

Val Asn Tyr Thr Ile Tyr Asp 430

Gly Phe Asn Leu Arg Asn Thr Asn

435 440

Ala Ala Asn Phe Asn Gly Gin 445

Asn Thr Glu Ile Asn Asn Met Asn

450 455

Thr Lys Leu Lys Asn Phe Thr 460

Gly Leu Phe Glu Phe Tyr Lys Leu 465 470

Cys Val Asp Gly Ile Ile Thr

475 480

Lys Thr Lys Cheese Leu Ile Glu 485

Arg Asn Lys Ala Leu Asn Asp 490 495

Leu Cys Ile Lys Val Asn Asn Trp 500

Leu Phe Ser Pro Cheese Glu SIO

Asp Asn Phe Thr Asn Asp Leu Asn

515 520

Gly Glu Glu Ile Thr Ser Asp 525

Thr Asn Ile Glu Ala Ala Glu Glu

530 335

Ile Cheese Leu Asp Leu Ile Gin 540

123

<td>Gin 545</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>Thr</td><td>The S50 faction</td><td>Asn</td><td>Phe</td><td>Asp</td><td colspan="2">Asn Glu 555</td><td>Pro</td><td>Glu</td><td>Asn</td><td>How much</td><td>Cheese 560</td>

<td>How much</td><td>Glu</td><td>Asn</td><td>Leu</td><td>Cheese 565</td><td>Cheese</td><td>Asp</td><td>How much</td><td>How much</td><td>Gly S70</td><td>Gin</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Underworld 575</td><td>Pro</td>

<td>Asn</td><td>How much</td><td>Glu</td><td>Arg ΞΒ0</td><td>Phe</td><td>Pro</td><td>Asn</td><td>Gly</td><td>Lys 5Θ5</td><td>List</td><td>Tyr</td><td>Glu</td><td>Leu</td><td>Asp S90</td><td>List</td><td>Tyr</td>

<td>Thr</td><td>Underworld</td><td>Phe 595</td><td>His</td><td>Tyr</td><td>Leu</td><td>Arg</td><td>Ala 600</td><td>Gin</td><td>Glu</td><td>Phe</td><td>Glu</td><td>His 605</td><td>Gly</td><td>List</td><td>Cheese</td>

<td>Arg</td><td>How much 610</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Asn</td><td>Cheese 615</td><td>Val</td><td>Asn</td><td>Glu</td><td>Ala</td><td>Leu 62 0</td><td>Leu</td><td>Asn</td><td>Pro</td><td>Cheese</td>

<td>Arg 625</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Phe</td><td>Phe 630</td><td>Cheese</td><td>Cheese</td><td>Asp</td><td>Tyr</td><td>Val 635</td><td>List</td><td>List</td><td>Val</td><td>Asn</td><td>Lys 640</td>

<td>Ala</td><td>Thr</td><td>Glu</td><td>Ala</td><td>Ala 645</td><td>Underworld</td><td>Phe</td><td>Leu</td><td>Gly</td><td>Trp 650</td><td>Val</td><td>Glu</td><td>Gin</td><td>Leu</td><td>Val 6 55</td><td>Tyr</td>

<td>Asp</td><td>Phe</td><td>Thr</td><td>Asp 660</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>Glu</td><td>Val 665</td><td>Cheese</td><td>Thr</td><td>Thr</td><td>Asp</td><td>Lys 670</td><td>How much</td><td>Ala</td>

<td>Asp</td><td>How much</td><td>Thr 675</td><td>How much</td><td>How much</td><td>How much</td><td>Pro</td><td>Tire 680</td><td>How much</td><td>Gly</td><td>Pro</td><td>Ala</td><td>Leu 685</td><td>Asn.</td><td>How much</td><td>Gly</td>

<td>Asn</td><td>Underworld 690</td><td>Leu</td><td>Tyr</td><td>List</td><td>Asp</td><td>Asp 695</td><td>Phe</td><td>Shaft</td><td>Gly</td><td>Ala</td><td>Leu 700</td><td>How much</td><td>Phe</td><td>Cheese</td><td>Gly</td>

<td>Ala 705</td><td>Val</td><td>How much</td><td>Leu</td><td>Leu</td><td>Glu 710</td><td>Phe</td><td>How much</td><td>Pro</td><td>Glu</td><td>How much 715</td><td>Ala</td><td>How much</td><td>Pro</td><td>Val</td><td>Leu 720</td>

<td>Gly</td><td>Thr</td><td>Phe</td><td>Ala</td><td>Leu 725</td><td>Val</td><td>Cheese</td><td>Tyr</td><td>How much</td><td>Ala 730</td><td>Asn</td><td>List</td><td>Val</td><td>Leu</td><td>Thr 735</td><td>Val</td>

<td>Gin</td><td>Thr</td><td>How much</td><td>Asp 740</td><td>Asn</td><td>Ala</td><td>Leu</td><td>Cheese</td><td>Lys 745</td><td>Arg</td><td>Asn</td><td>Glu</td><td>List</td><td>Trp 750</td><td>Asp</td><td>Glu</td>

<td>Val</td><td>Tyr</td><td>List 755</td><td>Tyr</td><td>How much</td><td>val</td><td>Thr</td><td>Asn 760</td><td>Trp</td><td>Leu</td><td>Ala</td><td>List</td><td>Val 765</td><td>Asn</td><td>Thr</td><td>Gin</td>

<td>How much</td><td>Asp 770</td><td>Leu</td><td>How much</td><td>Arg</td><td>List</td><td>Lys 775</td><td>Underworld</td><td>List</td><td>Glu</td><td>Ala</td><td>Leu 780</td><td>Glu</td><td>Asn</td><td>Gin</td><td>Ala</td>

124

Glu Ala Thr Lys Ala Ile Ile Asn 7Θ5 790

Gin Tyr Asn Gin Tyr Thr Glu

795 800

Glu Glu Lys Asn Asn Ile Asn Phe B05

Ile Asp Asp Leu Ser Ser Lys 810 815

Leu Asn Glu Ser Ile Asn Lys Ala 820

Ile Asn Ile Asn Lys Phe Leu 830

Asn Gin Cys Ser Val Ser Tyr Leu

835 840

Asn Ser Met Ile Pro Tyr Gly Θ45

Val Lys Arg Leu Glu Asp Phe Asp

850 855

Leu Lys Asp Ala Leu Leu cheese 880

Lys Tyr ile Tyr Asp Asn Arg Gly 065 870

Leu Ile Gly Gin Val Asp Arg

875 880

Leu Lys Asp Lys Val Asn Asn Thr 085

Thr Asp Ile Pro Phe Gin

890 895

Leu Ser Lys Tyr Val Asp Asn Gin

900

Leu Leu Ser Thr Leu Asp 910 &lt;210&gt; 17 &lt;211&gt; 2715 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 17

125

<td>ggatccgoes</td><td>tcatgccgat</td><td>caccatcaac</td><td>aacttcaact</td><td>acagcgatcc</td><td>ggtggataac</td><td> 60</td>

<td>aaaaacatcc</td><td>tgtacctgga</td><td>tacccatctg</td><td>aataccctgg</td><td>cgaacgaacc</td><td>ggaaaaagcg</td><td> 120</td>

<td>tttcgtatca</td><td>ccggcaacat</td><td>ttgggttatt</td><td>ccggatcgtt</td><td>ttagccgtaa</td><td>cagcaacccg</td><td> 100</td>

<td>aatctgaata</td><td>aaccgccgcg</td><td>tgttaccagc</td><td>ccgaaaagcg</td><td>gttattacga</td><td>taagaactat</td><td> 240</td>

<td>ctgagcaccg</td><td>atagcgataa</td><td>agataccttc</td><td>ctgaaagaaa</td><td>tcatcaaact</td><td>gttcaaacgc</td><td> 300</td>

<td>atcaacagcc</td><td>gtgaaattgg</td><td>cgaagaactg</td><td>atctatcgcc</td><td>tgagcaccga</td><td>tattccgttt</td><td> 360</td>

<td>ccgggcaaca</td><td>acaacacccc</td><td>gatcaacacc</td><td>tttgatttcg</td><td>atgtggattt</td><td>caacagcgtt</td><td> 420</td>

<td>gatgttaaaa</td><td>cccgccaggg</td><td>taacaattgg</td><td>gtgaaaaccg</td><td>gcagcattaa</td><td>cccgagcgtg</td><td> 460</td>

<td>attattaccg</td><td>gtccgcgcga</td><td>aaaeattatt</td><td>gatccggaaa</td><td>ccagcacctt</td><td>taaactgacc</td><td> 54 0</td>

<td>aacaacacct</td><td>ttgcggcgca</td><td>ggaaggtect</td><td>ggcgcgctga</td><td>gcattattag</td><td>cattagcccg</td><td> 600</td>

126

<td>cgctttatgc</td><td>tgacctatag</td><td>caacgcgacc aacgatgttg</td><td>gtgaaggccg</td><td>tttcagcaaa</td><td> 660</td>

<td>agcgaatttt</td><td>gcatggaccc</td><td>gatcctgatc ctgatgcatg</td><td>aactgaacca</td><td>tgcgatgcat</td><td> 720</td>

<td>aacctgtatg</td><td>catcgcgat</td><td>tccgaacgat cagaccatta</td><td>gcagcgtgaa</td><td>cagcaacatc</td><td>7B0</td>

<td>ttttacagca</td><td>agtacaacgt</td><td>gaaactggaa tatgcggaaa</td><td>tctatgcgtt</td><td>tggcagtccg</td><td> 34 0</td>

<td>accattgatc</td><td>tgattccgaa</td><td>aagcgcgcgc aaataettcg</td><td>aagaaaaagc</td><td>gctggattac</td><td> 900</td>

<td>tatcgcagca</td><td>ttgcgaaacg</td><td>tctgaacagc attachaccg</td><td>cgatccgag</td><td>cagcttcaac</td><td> 960</td>

<td>aaatatatcg</td><td>gcgaatataa</td><td>acagaaactg atccgcaaat</td><td>atcgctttgt</td><td>ggtggaaagc</td><td> 1020</td>

<td>agcggcgaag</td><td>ttaccgttaa</td><td>ccgcaataaa ttcgtggaac</td><td>tgtacaaega</td><td>actgacccag</td><td>ioao</td>

<td>atcttcaccg</td><td>aatttaacta</td><td>tgcgaaaatc tataacgtgc</td><td>agaaccgtaa</td><td>aatctacctg</td><td> 1140</td>

<td>agcaacgtgt</td><td>ataccccggt</td><td>gaccgcgt attctggatg</td><td>ataacgtgta</td><td>cgatatccag</td><td> 1200</td>

<td>aacggcttta</td><td>acatcccgaa</td><td>aagcaacctg aacgtcctgt</td><td>ttatgggcaa</td><td>gaacctgagc</td><td> 1260</td>

<td>cgtaatcegg</td><td>cgctgcgtaa</td><td>agtgaacccg gaaaacatgc</td><td>tgtacctgtt</td><td>caccaaattt</td><td> 1320</td>

<td>tgcgtcgacg</td><td>cgatagatgg</td><td>tagatttggc ggtttcacgg</td><td>gcgcacgcaa</td><td>atcagcgcgt</td><td> 1380</td>

<td>aaattagcta</td><td>accaggcgct</td><td>agcgggcggt ggcggtagcg</td><td>gcggtggcgg</td><td>tagcggcggt</td><td> 1440</td>

<td>ggcggtagcg</td><td>cactagtgct</td><td>gcagtgtcgt gaactgctgg</td><td>tgaaaaacac</td><td>cgatctgccg</td><td> 1800</td>

<td>tttattggcg</td><td>asacagcga</td><td>tgtgaaaacc gatatcttcc</td><td>tgcgcaaaga</td><td>tatcaacgaa</td><td> 1560</td>

<td>gaaaccgaag</td><td>tgatctacta</td><td>cccggataac gtgagcgttg</td><td>atcaggtgat</td><td>ccEgagcaaa</td><td> 1620</td>

<td>aacaccagcg</td><td>aacatggtca</td><td>gctggatctg ctgtatccga</td><td>gcattgatag</td><td>cgaaagcgaa</td><td> 1680</td>

<td>attętgacgg</td><td>gcgaaaacca</td><td>ggtgttttac gataaccgta</td><td>cccagaacgt</td><td>ggattacctg</td><td> 1740</td>

<td>aacagctatt</td><td>actacctgga</td><td>aagccagaaa ctgagcgata</td><td>acgtggaaga</td><td>ttttaccttt</td><td> 1800</td>

<td>acccgcagca</td><td>ttgaagaagc</td><td>gctggataac agcgcgaaag</td><td>tttacaccta</td><td>ttttccgacc</td><td> 1860</td>

<td>ctggcgaaca</td><td>aagttaatgc</td><td>gggtgttcag ggeggtctgt</td><td>ttetgatgtg</td><td>ggagaacgat</td><td> 1920</td>

<td>gtggtggaag</td><td>atttcaccac</td><td>caacatcctg cgtaaagata</td><td>ccctggataa</td><td>aatcagcgat</td><td> 1980</td>

<td>gttagcgcga</td><td>ttattccgta</td><td>tattggtccg gcgctgaaaa</td><td>ttagcaatag</td><td>cgtgcgtcgt</td><td> 2040</td>

<td>ggcaatttta</td><td>ccgaagcgtt</td><td>tgcggttacc ggtgtgacca</td><td>ttctgctgga</td><td>agcgtttccg</td><td> 2100</td>

<td>gaatttacca</td><td>ttccggcgct</td><td>gggtgcgttt gtgatctata</td><td>gcaaagtgca</td><td>ggaacgcaac</td><td> 2160</td>

<td>gaaatcatca</td><td>aaaccatcga</td><td>taactgcctg gaacagcgta</td><td>ttaaaagctg</td><td>gaaagatagc</td><td> 2220</td>

<td>tatgaatgga</td><td>Egatgggcac</td><td>ctggctgagc cgtattatca</td><td>cccagttcaa</td><td>caacatcagc</td><td> 2280</td>

<td>tacaagatgt</td><td>acgatagcct</td><td>gaactatcag gcgggtgcga</td><td>CCaaagcgaa</td><td>aatcgatctg</td><td> 2340</td>

gaatacaaaa aatacagcgg cagcgataaa gaaaacatca aaagacaggc tgaaaacctg 2400

127

<td>aaaaacagcc</td><td>tggatgtgaa</td><td>aattagcgaa</td><td>gcgaCgaata</td><td>acatcaacaa</td><td>attcatccgc</td><td> 2460</td>

<td>gaatgcagcg</td><td>tgacctacct</td><td>gttcaaaaac</td><td>atgctgccga</td><td>aagtgatcga</td><td>tgaactgaac</td><td> 2520</td>

<td>gttgatc</td><td>gcaacaccaa</td><td>agcgaaactg</td><td>atcaacctga</td><td>tcgatagcca</td><td>caacattatt</td><td> 2580</td>

<td>ctggtgggcg</td><td>aagtggataa</td><td>actgaaagcg</td><td>aaagttaaca</td><td>acagcttcca</td><td>gaacaccatc</td><td> 2640</td>

<td>ccgtttaaca</td><td>tcttcagcta</td><td>tacacacaac</td><td>agcctgctga</td><td>aagatatcat</td><td>caacgaatac</td><td> 2700</td>

<td>ttcaatctag</td><td>actag</td><td></td><td></td><td></td><td></td><td> 2715</td>

&lt;210&gt; 18 &lt;211&gt; 904 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt;

Synthetic &lt;400&gt; 18

128

Gly Ser Glu Phe Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp 15 1015

Pro Val Asp Asn lys Asn Ile Leu Tyr Lau Asp Thr His Leu Asn Thr 20 2530

Leu Ala Asn. Glu Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp 35 4045

Val Ile pro Asp Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys 50 5560

Pro Pro Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr 65 70 7580

Leu Ser Thr Asp Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys 85 909S

Leu Phe Lys Arg Ile Asn Cheese Arg Glu Ile Gly Glu Glu Leu Ile Tyr 100 105 110

Arg Leu Ser Thr Asp Ile Pro phe Pro Gly Asn Asn Asn Thr Pro Ile 115 120 125

Asn Thr phe Asp Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr

130 135140

Arg Gin Gly Asn Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val

145 iso 155160

129

Ile Ile Thr Gly Pro Arg Glu Asn 165

Ile Ile Asp Pro Glu Thr Ser Thr

170 175

Phe Lys Leu Thr Asn Asn Thr Phe

180

Ala Ala Gin Glu Gly Phe Gly Ala 185 190

Leu Cheese Ile Cheese Ile Cheese Pro

195 200

Arg Phe Met Leu Thr Tyr cheese Asn 205

Ala Thr Asn Asp Val Gly Glu Gly

210 215

Arg Phe Cheese Lys Cheese Glu Phe Cys

220

Met Asp Pro Ile Leu Ile Leu Met 225 230

His Glu Leu Asn His Ala Met His

235 240

Asn Leu Tyr Gly Background Ala Ile Pro 245

Asn Asp Gin Thr Ile Cheese Cheese Val

250 255

Thr Che Asn Ile Phe Tyr Ser Gin 260

Tyr Asn Val Lys Leu Glu Tyr Ala 265 270

Glu Ile Tyr Ala Phe Gly Gly Pro 275 2S0

Thr Ile Asp Leu Ile Pro Lys Ser 285

Ala Arg Lys Tyr Phe Glu Glu Lys

290 295

Ala Leu Asp Tyr. Tyr Arg Ser Ile

300

Ala Lys Arg Leu Asn Ser Ile Thr 305 310

Thr Ala Asn Pro Ser Ser Fhe Asn

315 320

Lys Tyr ile Gly Glu Tyr Lys Gin

325

Lys Leu Ile Arg Lys Tyr Arg Phe

330 335

Val Val Glu Cheese Gly Glu Val

340

Thr Val Asn Arg Asn Lys Phe Val 345 350

Glu Leu Tyr Asn Glu Leu Thr Gin

355 360

Ile Phe Thr Glu Phe Asn Tyr Ala 365

Lys ile Tyr Asn Val Gin Asn Arg

370 375

Lys Ile Tyr Leu Ser Asn val Tyr 380

Thr Pro Val Thr Ala Asn Ile Leu 385 390

Asp Asp Asn Val Tyr Asp Ile Gin

395 400

130

Asn Gly Phe Asn Ile Pro Lys Ser 405

Asn Leu Asn Val Leu Phe Met Gly

410 415

Gin Asn Leu Ser Arg Asn Pro Ala 420

Leu Arg Lys Val Asn Pro Glu Asn 425 430

Met Leu Tyr Leu Phe Thr Lys Phe

435 440

Cys Val Asp Ala Ile Asp Gly Arg

445

Phe Gly Gly Phe Thr Gly Ala Arg

450 455

Lys cheese Ala Arg Lys Leu Ala Asn 460

Gin Ala Leu Ala Gly Gly Gly Gly 465 470

Gly Gly Gly Gly Cheese Gly Gly Cheese

475 480

Gly Gly Cheese Ala Leu Val Leu Gin 485

Cys Arg Glu Leu Leu Val Lys Asn 490 495

Thr Asp Leu Pro Phe Ile Gly Asp 500 ile Ser Asp vai Lys Thr Asp Ile 505 510

Phe Leu Arg Lys Asp Ile Asn Glu

515 520

Glu Thr Glu Val Ile Tyr Tyr Pro 525

Asp Asn Val Ser Val Asp Gin Val 530 535

Ile Leu Cheese Lys Asn Thr Ser Glu

540

His Gly Gin Leu Asp Leu Leu Tyr 545 550

Pro Cheese Ile Asp Ser Glu Ser Glu

555 560

How much Leu. Pro Gly Glu Asn Gin Val 555

Phe Tyr Asp Asn Arg Thr Gin Asn 570 575

Val Asp Tyr Leu Asn Ser Tyr Tyr 580

Tyr Leu Glu Ser Gin Lys Leu Ser 585 590

Asp Asn Val Glu Asp Phe Thr Phe

595 600

Thr Arg Ser ile Glu Glu Ala Leu

605

Asp Asn Ser Ala Lys Val Tyr Thr

610 615

Tyr Phe Pro Thr Leu Ala Asn Lys

620

Val Asn Ala Gly Val Gin Gly Gly 62Ξ 630

Leu Phe Leu Met Trp Ala Asn Asp

635 640

131

<td>Val</td><td>Val</td><td>Glu</td><td>Asp</td><td>Phe 645</td><td>Thr</td><td>Thr</td><td>Asn</td><td>How much</td><td>Leu 650</td><td>Arg</td><td>List</td><td>Asp</td><td>Thr</td><td>Leu 655</td>

<td>List</td><td>How much</td><td>Cheese</td><td>Asp 650</td><td>Val</td><td>Cheese</td><td>Ala</td><td>Background</td><td>How much 665</td><td>Pro</td><td>Tyr</td><td>How much</td><td>Gly</td><td>Pro 670</td><td>Ala</td>

<td>Asn</td><td>How much</td><td>cheese 675</td><td>Asn</td><td>Cheese</td><td>val</td><td>Arg</td><td>Arg 580</td><td>Gly</td><td>Asn</td><td>Phe</td><td>Thr</td><td>G1U 685</td><td>Ala</td><td>Phe</td>

<td>Val</td><td>Thr 690</td><td>Gly</td><td>Val</td><td>Thr</td><td>How much</td><td>Leu 895</td><td>Leu</td><td>Glu</td><td>Ala</td><td>Phe</td><td>Pro 700</td><td>Glu</td><td>Phe</td><td>Thr</td>

<td>Pro 70S</td><td>Ala</td><td>Leu</td><td>Gly</td><td>Ala</td><td>Fhe 710</td><td>val</td><td>How much</td><td>Tyr</td><td>Cheese</td><td>Lys 715</td><td>or</td><td>Gin</td><td>Glu</td><td>Arg</td>

<td>Glu</td><td>How much</td><td>How much</td><td>List</td><td>Thr 725</td><td>How much</td><td>Asp</td><td>Asn</td><td>Cys</td><td>Leu 730</td><td>Glu</td><td>Gin</td><td>Arg</td><td>How much</td><td>Lys 735</td>

<td>Trp</td><td>List</td><td>Asp</td><td>Cheese 740</td><td>Tyr</td><td>Glu</td><td>Trp</td><td>Underworld</td><td>It 745</td><td>Gly</td><td>Thr</td><td>Trp</td><td>Leu</td><td>Cheese 7S0</td><td>Arg</td>

<td>How much</td><td>Thr</td><td>Gin 7SS</td><td>Phe</td><td>Asn</td><td>Asn</td><td>How much</td><td>Cheese 760</td><td>Tyr</td><td>Gin</td><td>Underworld</td><td>Tyr</td><td>Asp 755</td><td>Cheese</td><td>Leu</td>

<td>Tyr</td><td>Gin 770</td><td>Ala</td><td>Gly</td><td>Ala</td><td>How much</td><td>Lys 775</td><td>Ala</td><td>List</td><td>How much</td><td>Asp</td><td>Leu 780</td><td>Glu</td><td>Tyr</td><td>List</td>

<td>Tire 788</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>Asp</td><td>Lys 790</td><td>Glu</td><td>Asn</td><td>How much</td><td>List</td><td>Cheese 795</td><td>Gin</td><td>val</td><td>Glu</td><td>Asn</td>

<td>List</td><td>Asn</td><td>Cheese</td><td>Leu</td><td>Asp 805</td><td>Val</td><td>List</td><td>How much</td><td>Cheese</td><td>Glu 810</td><td>Ala</td><td>Underworld</td><td>Asn</td><td>Asn</td><td>How much 815</td>

<td>List</td><td>Phe</td><td>How much</td><td>Arg 820</td><td>Glu</td><td>Cys</td><td>Cheese</td><td>Val</td><td>Thr 825</td><td>Tyr</td><td>Leu</td><td>Phe</td><td>List</td><td>To 830</td><td>Underworld</td>

<td>Pro</td><td>List</td><td>Val 835</td><td>How much</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Asn 840</td><td>Glu</td><td>Phe</td><td>Asp</td><td>Arg</td><td>Asn 845</td><td>Thr</td><td>List</td>

<td>Lyg</td><td>Leu 880</td><td>How much</td><td>Asn</td><td>Leu</td><td>How much</td><td>Asp 855</td><td>Cheese</td><td>His</td><td>Asn</td><td>Background</td><td>Background 860</td><td>Leu</td><td>Val</td><td>Gly</td>

<td>Val 865</td><td>Asp</td><td>List</td><td>Leu</td><td>List</td><td>Ala 870</td><td>List</td><td>Val</td><td>Asn</td><td>Asn</td><td>Cheese 875</td><td>Phe</td><td>Gin</td><td>Asn</td><td>Thr</td>

<td>Pro</td><td>Phe</td><td>Asn</td><td>background</td><td>Phe</td><td>Cheese</td><td>Tire?</td><td>Thr</td><td>Asn</td><td>Asn</td><td>Cheese</td><td>Leu</td><td>Leu</td><td>List</td><td>Asp</td>

Asp

Leu

Ala

How much

Asn

720

A-9

How much

Asn.

List

Leu 800

Asn

Leu

Ala

Glu

How much 880

How much

132

835

890

395

Ile Asn Glu Tyr Phe Asn Leu Asp 900 &lt;210&gt; 19 &lt;211&gt; 2742 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 19

133

<td>ggatccgoes</td><td>tcatgccgat</td><td>caccatcaac</td><td>aacttcaact</td><td>acagcgatcc</td><td>ggtggataac</td><td> 60</td>

<td>aaaaacatcc</td><td>tgtacctgga</td><td>tacccatctg</td><td>aataccctgg</td><td>cgaacgaacc</td><td>ggaaaaagcg</td><td> 120</td>

<td>tttcgtatca</td><td>ccggcaacat</td><td>ttgggttatt</td><td>ccggatcgtt</td><td>ttagccgtaa</td><td>cagcaacccg</td><td> 180</td>

<td>aatctgaata</td><td>aaccgccgcg</td><td>tgttaccage</td><td>ccgaaaagcg</td><td>gttattacga</td><td>tccgaactat</td><td> 240</td>

<td>ctgagcaccg</td><td>atagcgataa</td><td>agataccttc</td><td>ctgaaagaaa</td><td>tcatcaaact</td><td>gttcaaacgc</td><td> 300</td>

<td>atcaacagcc</td><td>gtgaaattgg</td><td>cgaagaactg</td><td>atctatcgcc</td><td>tgagcaccga</td><td>tattccgttt</td><td> 360</td>

<td>ccgggcaaca</td><td>acaacacccc</td><td>gatcaacacc</td><td>tttgatttcg</td><td>atgtggattt</td><td>caacagcgtt</td><td> 420</td>

<td>gatgttaaaa</td><td>cccgccaggg</td><td>taacaattgg</td><td>gtgaaaaccg</td><td>gcagcattaa</td><td>cccgagcgtg</td><td> 480</td>

<td>attattaccg</td><td>gtccgcgcga</td><td>aaacattatt</td><td>gatccggaaa</td><td>ccagcacctt</td><td>taaactgacc</td><td> 540</td>

<td>aacaacacct</td><td>ttgcggcgca</td><td>ggaaggtttt</td><td>ggcgcgctga</td><td>gcattaEtag</td><td>cattagcccg</td><td> 600</td>

<td>cgctttatgc</td><td>tgacctatag</td><td>caacgcgacc</td><td>aacgatgttg</td><td>gtgaaggccg</td><td>tttcagcaaa</td><td> 660</td>

<td>agcgaatttt</td><td>gcatggaccc</td><td>gatcctgatc</td><td>ctgatgcatg</td><td>aactgaacca</td><td>tgcgatgcat</td><td> 720</td>

<td>aacctgtatg</td><td>catcgcgat</td><td>tccgaacgat</td><td>cagaccatta</td><td>gcagcgtgac</td><td>cagcaacatc</td><td> 780</td>

<td>ttttacagcc</td><td>agtacaacgt</td><td>gaaactggaa</td><td>tatgcggaaa</td><td>tctatgcgtt</td><td>tggcggtccg</td><td> 840</td>

<td>accattgatc</td><td>tgattccgaa</td><td>aagcgcgcgc</td><td>aaatacttcg</td><td>aagaaaaagc</td><td>gctggattac</td><td> 900</td>

<td>tatcgcagca</td><td>ttgcgaaacg</td><td>tctgaacagc</td><td>attachaccg</td><td>cgatccgag</td><td>cagcttcaac</td><td> 860</td>

<td>aaatatatcg</td><td>gcgaatataa</td><td>acagaaactg</td><td>atccgcaaat</td><td>atcgctttgt</td><td>ggtggaaagc</td><td> 1020</td>

<td>^and9cggcgaag</td><td>ttaccgttaa</td><td>ccgcaataaa</td><td>ttcgtggaac</td><td>tgtacaacga</td><td>actgacccag</td><td> 1080</td>

<td>atcttcaccg</td><td>aatttaacta</td><td>tgcgaaaatc</td><td>tataacgtgc</td><td>agaaccgtaa</td><td>aatctacctg</td><td> 1140</td>

<td>agcaacgtgt</td><td>ataccccggt</td><td>gaccgcgoes</td><td>attctggatg</td><td>ataacgtgta</td><td>cgatatccag</td><td> 1200</td>

<td>aacggcttta</td><td>acatcccgaa</td><td>aagcaacctg</td><td>aacgttctgt</td><td>ttatgggcca</td><td>gaacctgagc</td><td> 1260</td>

<td>cgtaatccgg</td><td>agctgcgtaa</td><td>agtgaacccg</td><td>gaaaacatgc</td><td>tgtacctgtt</td><td>caccaaattt</td><td> 1320</td>

<td>tgcgtcgacg</td><td>gcatcattac</td><td>ctccaaaact</td><td>aaatctctga</td><td>distributor</td><td>atttggcggt</td><td> 1380</td>

134

<td>ttcacgggcg</td><td>cacgcaaatc</td><td>agcgcgtaaa</td><td>ttagctaacc</td><td>aggcgctagc</td><td>gggcggtggc</td><td> 1440</td>

<td>ggtagcggcg</td><td>gtggcggtag</td><td>&lt;= 9gcggtggc</td><td>ggtagcgcac</td><td>CagCgctgca</td><td>gtgtcgtgaa</td><td> 1500</td>

<td>ctgctggtga</td><td>aaaacaccga</td><td>tctgccgttt</td><td>attggcgata</td><td>tcagcgatgt</td><td>gaaaaccgat</td><td> 1560</td>

<td>atcttcctgc</td><td>gcaaagatat</td><td>your plastic</td><td>accgaagtga</td><td>tctactaccc</td><td>ggataacgtg</td><td> 1620</td>

<td>agcgttgatc</td><td>aggtgatcct</td><td>gagcaaaaac</td><td>accagcgaac</td><td>atggtcagct</td><td>ggatctgctg</td><td> 1680</td>

<td>tatccgagca</td><td>ttgatagcga</td><td>aagcgaaatt</td><td>ctgccgggcg</td><td>aaaaccaggt</td><td>gttttacgat</td><td> 1740</td>

<td>aaccgtaccc</td><td>agaacgtgga</td><td>ttacctgaac</td><td>agctattact</td><td>acctggaaag</td><td>ccagaaactg</td><td> 1800</td>

<td>agcgataacg</td><td>tggaagattt</td><td>tacctttacc</td><td>cgcagcattg</td><td>aagaagcgct</td><td>ggataacagc</td><td>1B60</td>

<td>gcgaaagttt</td><td>acacctattt</td><td>tccgaccctg</td><td>gcgaacaaag</td><td>ttaatgcggg</td><td>tgttcagggc</td><td> 1920</td>

<td>ggtctgtttc</td><td>tgatgtgggc</td><td>gaacgatgtg</td><td>gtggaagatt</td><td>tcaccaccaa</td><td>catcctgcgt</td><td> 1980</td>

<td>aaagataccc</td><td>tggataaaat</td><td>cagcgatgtt</td><td>agcgcgatca</td><td>ttccgtatat</td><td>tggtccggcg</td><td> 2040</td>

<td>ctgaacatta</td><td>gcaatagcgt</td><td>gcgtcgtggc</td><td>aattttaccg</td><td>aagcgtttgc</td><td>ggttaccggt</td><td> 2100</td>

<td>gtgaccattc</td><td>tgctggaagc</td><td>gtttccggaa</td><td>tttaccattc</td><td>cggcgctggg</td><td>tgcgtttgtg</td><td> 2160</td>

<td>atctatagca</td><td>aagtgcagga</td><td>acgcaacgaa</td><td>atcatcaaaa</td><td>ccatcgataa</td><td>ctgcctggaa</td><td> 2220</td>

<td>cagcgtatta</td><td>aacgctggaa</td><td>agatagctate</td><td>gaatggatga</td><td>tgggcacctg</td><td>gctgagccgt</td><td> 2280</td>

<td>attaEcaccc</td><td>agttcaacaa</td><td>catcagctac</td><td>cagatgtacg</td><td>atagcctgaa</td><td>ctatcaggcg</td><td> 2340</td>

<td>ggtgcgatta</td><td>aagcgaaaat</td><td>cgatctggaa</td><td>tacaaaaaat</td><td>acagcggcag</td><td>agataaagaa</td><td> 2400</td>

<td>aacatcaaaa</td><td>gccaggttga</td><td>aaacctgaaa</td><td>aacagcctgg</td><td>atgtgaaaat</td><td>tagagaagcg</td><td> 2460</td>

<td>atgaataaea</td><td>tcaacaaatt</td><td>catccgcgaa</td><td>tgcagcgtga</td><td>cctacctgtt</td><td>caaaaacatg</td><td> 2520</td>

<td>ctgccgaaag</td><td>tgatcgatga</td><td>acbgaacgaa</td><td>tttgatcgca</td><td>acaccaaagc</td><td>gaaactgatc</td><td> 2580</td>

<td>aacctgatcg</td><td>atagccacaa</td><td>cattattctg</td><td>gtgggcgaag</td><td>tggataaact</td><td>gaaagcgaaa</td><td> 2640</td>

<td>gttaacaaca</td><td>gcttccagaa</td><td>caccatcccg</td><td>tttaacatct</td><td>tcagctatac</td><td>caacaacagc</td><td> 2700</td>

<td>ctgctgaaag</td><td>atatcatcaa</td><td>cgaacttc</td><td>aatctagact</td><td>ag</td><td></td><td> 2742</td>

&lt;210&gt; 20 &lt;211&gt; 913 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 20

135

Gly ser Glu Phe Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp ¹ S 10 IS

136

Pro Val

Asp Asn 20

Lys Asn

Leu Ala

Asn Glu 35

Pro Glu

With Val

Pro Asp

Arg Phe

Pro Pro 65

Arg Val

Thr Ser 70

Leu Ser

Thr Asp

Asp cheese 85

Leu Phe

Lys Arg 100

How many Asn

How much Leu

Lys Ala 40

Arg cheese 55

Pro Lys

Lys Asp

Arg cheese

Tire of Leu 25

Phe Arg

Asn Ser

Gly cheese

Thr Phe

Glu Background 105

Arg Leu

Thr Cheese 115

Asp He

Pro Faction

120

Pro Gly

Asp Thr

How Much Thr

Asn Pro

Tire 75

Leu Lys

Gly Glu

Abtl Asn

Asn Thr

130

Phe Asp

Phe Asp

Val Asp 135

Phe Asn

Val cheese

0

His Leu

Gly Asn 45

Asn Leu

Asp Pro

With Glu

Glu Leu

110

Asn Thr 125

Asp Val

Asn Thr

1le Trp

Asn Lys

Asn Tyr

How many Lys 95

How many Tyr

With Pro

Lys Thr

Arg Gin 145

Gly Asn

Asn Trp 150

Val Lys

Thr Gly

With cheese 155

Asn Pro

Cheese val

160

How many How many

Thr Gly

Pro Arg 165

Glu Asn

How many How many

170

Asp Pro

Glu Thr

Thr Cheese 175

Phe Lys

Leu Thr

1B0

Asn Asn

Thr Phe

Ala Ala 185

Gin Glu

Gly Phe 190

Gly Ala

Leu Ser

How much How much 195

With cheese

Pro cheese

200

Arg Phe

With Leu

Thr Tyr 205

Asn cheese

Ala Thr

210

Asn Asp

Val Gly

Glu Gly 215

Arg Phe

Lys cheese

220

Glu cheese

Phe Cys

Met Asp 22S

With Pro

Leu Ile

230

Leu Met

His Glu

Leu Asn 235

His Ala

Met His

240

Asn Leu

Tyr Gly

How Much Ala 245

Pro background

Asn Asp 250

Gin Thr how much cheese

Val 255 cheese

137

Thr Che Asn Background Phe Tyr Ser Gin Tyr 260 265

Glu Ile Tyr Ala Phe Gly Gly Pro Thr 275 200

Asn Val Lys Leu Glu Tyr Ala 270

Ile Asp Leu Ile Pro Lys Ser

205

Ala Arg Lys Tyr 290

Phe Glu Glu Lys

95

Ala Leu Asp Tyr Tyr Arg Ser Ile 300

Ala

305

Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser

310 315

Phe Asn

320

Lys Tyr how many Gly

Glu Tyr Lys Gin Lys Leu 325 330

Val Val Glu Ser

340

Gly Glu Val Thr Val Cheese

345

Glu Leu Tyr Asn 355

With Glu Leu Thr Gin

360

Phe Thr

How Much Arg Lys Tyr Arg Phe 335

Asn Arg Asn Lys Phe Val 350

Glu Phe Asn Tyr Ala 365

List

How many Tyr Asn Val Gin Asn Arg Lys 370 3? 5

Ile Tyr Leu Ser Asn Val Tyr

360

Thr Pro val Thr Ala Asn Background Leu 305 390

Asp Asp Asn Val Tyr asp Ile Gin

395 400

Asn Gly Phe Asn Ile Pro Lys Ser

405

Asn Leu Asn Val Leu Phe Met Gly 410 415

Gin Asn Leu Ser- Arg Asn Pro Ala 420

Leu Arg Lys Val Asn Pro Glu Asn 425 430

Met Leu Tyr Leu Phe Thr Lys Phe

435 440

Cys Val Asp Gly Ile Ile Thr Ser

445

Lys. Thr Lys Cheese Leu Ile 450

Glu Gly Arg Phe Gly 455

Gly Phe Thr Gly Ala 460

Arg Lys Ser Ala Arg Lys 465 470

Leu Ala Asn Gin Ala

475

Leu Ala Gly Gly Gly

480

Gly Ser Gly Gly Gly Gly 465

Gly Gly Gly Gly cheese

490

Ala Leu Val Leu cheese

495

Gin Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro Phe Ile Gly

138

How many Asn

500 505 510

With Asp

Glu Glu

530 545 with val

Tyr Pro

Asp 515 cheese

Thr Glu

Leu Ser

With cheese

Val Lys

With Val

Lys Asn

550

Asp Ser 565

Val Phe

Tire Asp

590

Asn Arg

Tire Tire

Leu Glu 595

Gin cheese

Phe Thr

610

Arg Ser

How much Glu

Thr Asp

520

Tyr Tyr S35

Thr Ser

Glu Ser

How Much Phe

Leu Arg

Pro Asp

Glu His

With Glu

570

Asn Val

540

Gly Gin 555

Leu Pro

Lys Asp 525

Val cheese

Leu Asp

Gly Glu

Asp Gin

Leu Leu

560

Asn Gin 575

Thr Tyr 635

The Pro faction

Thr Leu

630

Gly Leu

Phe Leu

Met Trp 645

With Asn

Leu Arg

660

Lys Asp

Thr Gin

Lily Leu 600

Glu Ala 615

Ala Asn

Al a Asn

Thr Leu

Asn Val 5S5

Asp cheese

Leu Asp

Asp Tyr

Leu Asn

590

Cheese Tyr

Asn Val

Asn Ser

620

How many How many

Pro Tyr

675

How much Gly

Pro Ala

680

Glu Asp 605

Ala Lys

Phe Thr

Val Tyr

Arg Gly 690

Asn Phe

Leu Glu 705

Ala phe

How many Tyr

Lys cheese

Asn Cys

Leu Glu

740

Thr Glu

Pro Glu 710

Val Gin 725

Gin Arg

Ala phe 695

Phe Thr

Glu Arg

How many Lys

Light Val

Asp Val 650

Asp Lys 665

Leu Asn

Ala Val

How much Pro

Asn Glu

730

Arg Trp 745

Asn Ala 635

Val Glu

How much Cheese

How much Cheese

Thr Gly

700

Ala Leu 715

How many How many

Gly Val

Gin Gly

640

Asp Phe

Asp Val

670

Asn Ser 685

Val Thr

Gly Ala

Lys Thr

Cheese Tyr

750

Thr Thr 655

Ala cheese

Val Arg

How much Leu

Phe Val

720

How many Asp 735

Glu Trp

Lys Asp

139

Met Met Gly Thr Trp Leu Ser Arg Ile Ile

755760

Thr Gin Phe Asn Asn Ile 765

Cheese Tyr Gin Met Tyr Asp Cheese Leu Asn Tyr Gin Ala Gly Ala Ile Lys

770 775760

Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu

785 790 795800

Asn Ile Lys Ser Gin Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys

805 810815

Ile Cheese Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser

820 825830

Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu

835 840845

Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn Leu Ile Asp 850 855860

Ser His Asn Ile Leu Val Gly Glu Val Asp Lys Leu Lys Ala Lys

865 870 875880

Val Asn Asn Ser Phe Gin Asn Thr Ile Pro Phe Asn Ile Phe Ser Tyr

885 890895

Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Leu 900 905910

Asp &lt;210&gt; 21 &lt;211&gt; 2673 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 21 ggatccatgg agttcgttaa caaacagttc aactataaag acccagttaa cggtgttgac 60 attgcttaca tcaaaatccc gaacgctggc cagatgcagc cggtaaaggc attcaaaattta cggtaaaggc attcaaaatcatctgtacgaaatcaccggtaaagaccaccgtacgaaattc 120 ccgtaaaatcaccgtacgaaattc 120cc gattaaatttacc

180

140

<td>ctgaacccgc</td><td>caccggaagc</td><td>gaaacaggtg</td><td>ccggtatctt</td><td>actatgactc</td><td>cacctacctg</td><td> 240</td>

<td>tctaccgata</td><td>acgaaaagga</td><td>caactacccg</td><td>aaaggtgtta</td><td>ccaaactgtt</td><td>cgagcgtatt</td><td> 300</td>

<td>tactccaccg</td><td>acctgggccg</td><td>tatgctgctg</td><td>actagcatcg</td><td>ttcgcggtat</td><td>cccgttctgg</td><td> 360</td>

<td>ggcggttcta</td><td>ccatcgaeac</td><td>cgaactgaaa</td><td>gtaatcgaca</td><td>ctaactgcat</td><td>caacgttatt</td><td> 420</td>

<td>cagccggacg</td><td>gttcctatcg</td><td>ttccgaagaa</td><td>ctgaacctgg</td><td>tgatcatcgg</td><td>cccgtctgct</td><td> 480</td>

<td>gatatcatcc</td><td>agttcgagtg</td><td>taagagcttt</td><td>ggtcacgaag</td><td>ttctgaacct</td><td>cacccgtaac</td><td> 540</td>

<td>ggctacggtt</td><td>ccactcagta</td><td>catccgtttc</td><td>tctccggact</td><td>tcaccttcgg</td><td>ttttgaagaa</td><td> 600</td>

<td>tccctggaag</td><td>tagacacgaa</td><td>cccactgctg</td><td>ggcgatggta</td><td>aattcgcaac</td><td>tgaccctgag</td><td> 660</td>

<td>gttaccctgg</td><td>ctcacgaact</td><td>gattcatgca</td><td>ggccaccgcc</td><td>tgtacggtat</td><td>cgccatcaat</td><td> 720</td>

<td>ccgaaccgtg</td><td>tcttcaaagt</td><td>taacaccaac</td><td>gcgtattacg</td><td>agatgtccgg</td><td>tctggaagtt</td><td> 780</td>

<td>agcttcgaag</td><td>aactgcgtac</td><td>ttttggcggt</td><td>cacgacgcta</td><td>aattcatcga</td><td>ctctctgcaa</td><td> 840</td>

<td>gaaaacgagt</td><td>tccgtctgta</td><td>ctactataac</td><td>aagttcaaag</td><td>atatcgaatc</td><td>caccctgaac</td><td> 900</td>

<td>aaagcgaaat</td><td>ccatcgtggg</td><td>taccactgct</td><td>tctctccagt</td><td>acatgaagaa</td><td>cgtttttaaa</td><td> 960</td>

<td>gaaaaatacc</td><td>tgctcagcga</td><td>agacacctcc</td><td>ggcaaattct</td><td>ctgtagacaa</td><td>gttgaaattc</td><td> 1020</td>

<td>gataaacttt</td><td>acaaaatgct</td><td>gactgaaatt</td><td>tacaccgaag</td><td>acaacttcgt</td><td>taagttcttt</td><td> 1080</td>

<td>aaagttctga</td><td>accgcaaaac</td><td>ctatctgaac</td><td>ttcgacaagg</td><td>cagtattcaa</td><td>aatcaacatc</td><td> 114 0</td>

<td>gtgccgaaag</td><td>ttaactacac</td><td>tatctacgat</td><td>ggtttcaacc</td><td>tgcgtaacac</td><td>caacctggct</td><td> 1200</td>

<td>gctaatttta</td><td>acggccagaa</td><td>cacggaaatc</td><td>aacaacatga</td><td>acttcacaaa</td><td>actgaaaaac</td><td> 1260</td>

<td>ttcactggtc</td><td>tgttcgagtt</td><td>ttacaagctg</td><td>ctgtgcgtcg</td><td>acggcatcat</td><td>tacctccaaa</td><td> 1320</td>

<td>actaaatctc</td><td>tgatagaagg</td><td>tagatacggt</td><td>ggtttcatgg</td><td>cgctagcggg</td><td>cggtggcggt</td><td> 1380</td>

<td>agcggcggtg</td><td>gcggtagcgg</td><td>cggtggcggt</td><td>agcgcactag</td><td>tgctgcagtg</td><td>tatcaaggtt</td><td> 1440</td>

<td>aacaactggg</td><td>atttattctt</td><td>cagcccgagt</td><td>gaagacaact</td><td>tcaccaacga</td><td>cctgaacaaa</td><td> 1500</td>

<td>ggtgaagaaa</td><td>tcacetcaga</td><td>tactaacatc</td><td>gaagcagccg</td><td>aagaaaacat</td><td>ctogctggac</td><td> 1560</td>

<td>ctgatccagc</td><td>agtactacct</td><td>gaactttaat</td><td>ttcgacaacg</td><td>agccggaaaa</td><td>catttctatc</td><td> 1620</td>

<td>gaaaacctga</td><td>gctctgatat</td><td>catcggccag</td><td>ctggaactga</td><td>tgccgaacat</td><td>cgaacgtttc</td><td> 1680</td>

<td>ccaaacggta</td><td>aaaagtacga</td><td>gctggacaaa</td><td>tataccatgt</td><td>tccaetacct</td><td>gcgcgcgcag</td><td> 1740</td>

<td>gttgaac</td><td>acggcaaatc</td><td>ccgtatcgca</td><td>ctgactaacE</td><td>ccgttaacga</td><td>agctctgctc</td><td> 1800</td>

<td>aacccgtccc</td><td>gtgtatacac</td><td>cttcttctct</td><td>agcgactacg</td><td>tgaaaaaggt</td><td>caacaaagcg</td><td> 1860</td>

<td>actgaagctg</td><td>caatgttctt</td><td>gggttgggtt</td><td>gaacagcttg</td><td>tttatgattt</td><td>taccgacgag</td><td> 1920</td>

<td>acgtccggag</td><td>tatetactac</td><td>cgacaaaatt</td><td>gcggatatca</td><td>ctatcatcat</td><td>cccgtacatc</td><td> 1980</td>

<td>ggtccggctc</td><td>tgaacattgg</td><td>caacatgctg</td><td>tacaaagacg</td><td>acttcgttgg</td><td>cgcactgatc</td><td> 2040</td>

141

<td>ttctccggtg</td><td>cggtgatcct</td><td>gctggagttc</td><td>atcccggaaa</td><td>tcgccatccc</td><td>ggtaętgggc</td><td> 2100</td>

<td>acctttgctc</td><td>tggtttctta</td><td>cattgcaaac</td><td>aaggttctga</td><td>ctgtacaaac</td><td>catcgacaac</td><td> 2160</td>

<td>gcgctgagca</td><td>aacgtaacga</td><td>aaaatgggat</td><td>gaagtttaca</td><td>aatatategt</td><td>gaccaactgg</td><td> 2220</td>

<td>ctggctaagg</td><td>ttaatactca</td><td>gatcgacctc</td><td>atccgcaaaa</td><td>aaatgaaaga</td><td>agcactggaa</td><td> 2280</td>

<td>aaccaggcgg</td><td>aagctaccaa</td><td>ggcaatcatt</td><td>aactaccagt</td><td>in cash</td><td>caccgaggaa</td><td> 2340</td>

<td>gaaaaaaaca</td><td>acatcaactt</td><td>caacatcgac</td><td>gatctgtcct</td><td>ctaaactgaa</td><td>cgatccatc</td><td> 2400</td>

<td>aacaaagcta</td><td>tgatcaacat</td><td>caacaagttc</td><td>ctgaaccagt</td><td>gctgtaag</td><td>ctatctgatg</td><td> 2460</td>

<td>aactccatga</td><td>tcccgtacgg</td><td>tgttaaacgt</td><td>ctggaggact</td><td>tcgatgcgtc</td><td>tctgaaagac</td><td> 2520</td>

<td>gccctgctga</td><td>aatacattta</td><td>cgacaaccgt</td><td>ggcactctga</td><td>tcggtcaggt</td><td>tgatcgtctg</td><td> 2580</td>

<td>aaggacaaag</td><td>tgaaeaatac</td><td>heptategaco</td><td>gacatccctt</td><td>ttcagctcag</td><td>taaatatgtc</td><td> 2640</td>

<td>gataaccaac</td><td>gccttttgtc</td><td>cactctagac</td><td>tag</td><td></td><td></td><td> 2673</td>

&lt;210&gt; 22 &lt;211&gt; 890 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 22

142

<td>Gly 1</td><td>Cheese Met</td><td>Glu</td><td>Phe Val Asn Lys 5</td><td>Gin Phe Asn 10</td><td>Tyr</td><td>List</td><td>Asp</td><td>Pro Val 15</td>

<td>Asn</td><td>Gly val</td><td>Asp 20</td><td>With Ala Tyr</td><td>Lys Ile Pro 25</td><td>Asn</td><td>Ala</td><td>Gly 30</td><td>Gin Met</td>

<td>Gin</td><td>Pro Val. 35</td><td>List</td><td>Ala Phe Lys Ile 40</td><td>His Asn Lys</td><td>How much</td><td>Trp 45</td><td>Val</td><td>How much Pro</td>

<td>GLU</td><td>Arg Asp 50</td><td>Thr</td><td>Phe Thr Asn Pro 55</td><td>Glu Glu Gly</td><td>Asp 60</td><td>Leu</td><td>Asn</td><td>Pro Pro</td>

<td>Pro 65</td><td>Glu Ala</td><td>List</td><td>Gin Val Pro Val 70</td><td>Tyr Tyr cheese 75</td><td>Asp</td><td>Cheese</td><td>Thr</td><td>Shoot Leo 80</td>

<td>Cheese</td><td>Thr Asp</td><td>Asn</td><td>Glu Lys Asp Asn 85</td><td>Tyr Leu Lys 90</td><td>Gly</td><td>Val</td><td>Thr</td><td>Lily Leu 95</td>

<td>Phe</td><td>Glu Arg</td><td>How much 100</td><td>Tyr Ser Thr Asp</td><td>Leu Gly Arg 105</td><td>Underworld</td><td>Leu</td><td>Leu 110</td><td>Thr Ser</td>

143

Gly Ser Thr Ile Asp Thr Glu

125

<td>He</td><td>Val</td><td>Arg 115</td><td>Gly</td><td>how much</td><td>Pro</td><td>Phe</td><td>Trp 120</td>

<td>Leu</td><td>Lys 130</td><td>Val</td><td>How much</td><td>ASp</td><td>Thr</td><td>Asn 13S</td><td>Cys</td>

<td>Cheese 145</td><td>Tyr</td><td colspan="2">Arg ^ Ser</td><td>_Glu</td><td>Glu 150</td><td>Leu</td><td>Asn</td>

<td>Asp</td><td>How much</td><td>How much</td><td>Gin</td><td>Phe 165</td><td>G1U</td><td>Cys</td><td>List</td>

<td>Leu</td><td>Thr</td><td>Arg</td><td>Asn 180</td><td>Gly</td><td>Tyr</td><td>Gly</td><td>Cheese</td>

<td>ASp</td><td>Phe</td><td>Thr 195</td><td>Phe</td><td>Gly</td><td>Phe</td><td>Glu</td><td>Glu 200</td>

<td>Leu</td><td>Leu 210</td><td>Gly</td><td>Ala</td><td>Gly</td><td>List</td><td>Phe 215</td><td>Ala</td>

<td>His 225</td><td>Glu</td><td>Leu</td><td>How much</td><td>His</td><td>Ala 230</td><td>Gly</td><td>His</td>

<td>Pro</td><td>Asn</td><td>Arg</td><td>Val</td><td>Phe 245</td><td>List</td><td>Val</td><td>Asn</td>

<td>Gly</td><td>Leu</td><td>Glu</td><td>Val 260</td><td>Cheese</td><td>Phe</td><td>Glu</td><td>Glu</td>

<td>Ala</td><td>List</td><td>Phe 275</td><td>How much</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Gin 260</td>

<td>Tyr</td><td>Asn 290</td><td>List</td><td>Phe</td><td>List</td><td>Asp</td><td>How much 295</td><td>Ala</td>

<td>How much 305</td><td>Val</td><td>Gly</td><td>Thr</td><td>Thr</td><td>Ala 310</td><td>Cheese</td><td>Leu</td>

<td>Glu</td><td>List</td><td>Tyr</td><td>Leu</td><td>Leu 325</td><td>Cheese</td><td>Glu</td><td>Asp</td>

<td>List</td><td>Leu</td><td>List</td><td>Phe 340</td><td>Asp</td><td>List</td><td>Leu</td><td>Tyr</td>

Asn Val Ile Gin Pro Asp Gly 140

Get Gly Pro Ser with Val

IBS 160

Phe Gly His Glu Val Leu Asn 170 175

Gin Tyr Ile Arg Phe Cheese Pro 190

Leu Glu Val Asp Thr Asn Pro 2 05

Asp Pro Ala Val Thr Leu Ala

220

Leu Tyr Gly how many Ala Ile Asn

235 240

Asn Ala Tyr Tyr Glu Met Ser

250 255

Arg Thr Phe Gly Gly His Asp 270

Asn Glu Phe Arg Leu Tyr Tyr '285

Thr Leu Asn Lys Ala Lys Ser

300

Tyr Met Lys Asn Val Phe Lys

315 320

Gly Lys Phe Ser Val Asp 330 335

Met Leu Thr Glu Ile Tyr Thr

350

144

Glu Asp

Leu Asn

370

Asn Tyr 3nS

Ala Asn

Lilies leu

Val Asp

Tyr Gly 450

Gly Cheese 465

Asn Asn

Asp Leu

Ala Glu

Phe Asn

530

Asn Phe 355

Phe Asp

Thr Ile

Phe Asn

Lys Asn 420

Gly Ile 435

Gly Phe

Gly Gly

Trp Asp

Asn Lys 500

Glu Asn 515

Phe Asp val Lys

Lys Ala

Tyr Asp 390

Gly Gin 405

Phe Thr

How Much Thr

Met Ala

Gly Gly 470

Leu Phe 405

Gly Glu

How much Cheese

Asn Glu

Phe Phe 360

Val Phe 375

Gly Phe

Asn Thr

Gly Leu

Lys 440 cheese

Leu Ala 455

Ala cheese

Phe Ser

With Glu

Leu Asp 520

Pro Glu 535

Light Val

Lys Background

Asn Leu

With Glu

410

The Glu 425

Thr Lys

Gly Gly

Leu Val

Prc Ser

490

Thr Ser 505

Leu Ile

With Asn

Leu Asn

Asn with 380

Arg Asn 395

Asn Asn

Phe Tyr

Leu cheese

Gly Gly 460

Leu Gin 475

Glu Asp

Asp Thr

Gin Gin

With cheese

540

Arg Lys 365

Val Pro

Thr Asn

Met Asn

Lily Leu 430

How Much Glu 445

Gly cheese

Cys Ile

Asn Phe

Asn Background

510

Tire 525

Glu Asn

Thr Tyr

Light Val

Leu Ala 400

Phe Thr 415

Leu Cys

Gly Arg

Gly Gly

Lys Val 480

Thr Asn 495

Glu Ala

Leu Thr

Leu Ser

<td>Cheese. 545</td><td>Asp</td><td>How much</td><td>How much</td><td>Gly</td><td>Gin 550</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Underworld</td>

<td>Pro</td><td>Asn</td><td>Gly</td><td>List</td><td>Lys 565</td><td>Tyr</td><td>Glu</td><td>Leu</td><td>Asp</td><td>Lys 570</td>

<td>Leu</td><td>Arg</td><td>Ala</td><td>Gin 580</td><td>Glu</td><td>Phe</td><td>Glu</td><td>His</td><td>Gly 585</td><td>List</td>

Pro Asn Ile Glu Arg Phe 555 560

Tyr Thr Met Phe His Tyr 575

Arg Ile Ala Leu Thr cheese 590

Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe

145

595 600

Phe Ser Set Asp Tyr Val Lys Lye

610 615

Met Phe Leu Gly Trp Val Glu Gin

625 630

Thr Ser Glu Val Ser Thr Thr Asp

645

Ile Pro Tyr Ile Gly Pro Ala Leu 660

Asp Asp Phe Val Gly Ala Leu Ile

675 680

With Glu Phe With Pro With Glu With Ala

690 695 vai Ser Tyr Ile Ala Asn Lye val 705 710

Ala Leu Ser Lys Arg Asn Glu Lys 725

Val Thr Asn Trp Leu Ala Lys Val 740

Lys Lys Met Lys Glu Ala Leu Glu

755 760

Ile Ile Asn Tyr Gin Tyr Asn Gin

770 775

Ile Asn Phe Asn Ile Asp Asp Leu 785 790

Asn Lye Ala Met Ile Αβη With Asn 805

Cheese Tyr Leu With Asn Cheese With Ile 820

Asp Phe Asp Ala Ser Leu Lys Asp

835 840

<td colspan="7"> 605</td>

<td>Asn</td><td>List</td><td>Ala 620</td><td>Thr</td><td>Glu</td><td>Ala</td><td>Ala</td>

<td>Val</td><td>Tire 635</td><td>Asp</td><td>Phe</td><td>Thr</td><td>Asp</td><td>Glu 640</td>

<td>How much 650</td><td>Ala</td><td>Asp</td><td>How much</td><td>Thr</td><td>How much 6 55</td><td>How much</td>

<td>How much</td><td>Gly</td><td>Α5Π</td><td>Underworld</td><td>Leu 670</td><td>Tyr</td><td>List</td>

<td>Cheese</td><td>Gly</td><td>Ala</td><td>or 685</td><td>How much</td><td>Leu</td><td>Leu</td>

<td>Val</td><td>Leu</td><td>Gly 700</td><td>Thr</td><td>Phe</td><td>Ala</td><td>Leu</td>

<td>Thr</td><td>Val 715</td><td>Gin</td><td>Thr</td><td>How much</td><td>Asp</td><td>Asn 720</td>

<td>Asp 730</td><td>Glu</td><td>Val</td><td>Tyr</td><td>List</td><td>Tyr 735</td><td>How much</td>

<td>Thr</td><td>Gin</td><td>How much</td><td>Asp</td><td>Leu 750</td><td>How much</td><td>Arg</td>

<td>Gin</td><td>Ala</td><td>Glu</td><td>Ala 765</td><td>Thr</td><td>List</td><td>Ala</td>

<td>Thr</td><td>Glu</td><td>Glu 780</td><td>Glu</td><td>List</td><td>Asn</td><td>Asn</td>

<td>Cheese</td><td>Lys 795</td><td>Leu</td><td>Asn</td><td>Glu</td><td>Cheese</td><td>How much 800</td>

<td>Phe 810</td><td>Leu</td><td>Asn</td><td>Gin</td><td>Cys</td><td>Cheese 815</td><td>Val</td>

<td>Tyr</td><td>Gly</td><td>Val</td><td>List</td><td>Arg 830</td><td>Leu</td><td>G1U</td>

<td>Leu</td><td>Leu</td><td>List</td><td>Tire 845</td><td>How much</td><td>Tyr</td><td>Asp</td>

146

Asn Arg Gly Thr 850

Leu Ile Gly Gin BS5

Val Asp Arg Leu Lys Asp Lys Val 860

Asn Asn Thr Leu 865

Ser Thr Asp 870 Nov Pro Phe Gin Leu Ser Lys Tyr Val

875 880

Asp Asn Gin Arg Leu Leu Ser Thr 885

Leu Asp

890 &lt;210&gt; 23 &lt;211&gt; 2751 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 23

147

<td>ggatccatgg</td><td>agttcgttaa</td><td>caascagttc</td><td>aactataaag</td><td>acccagttaa</td><td>cggtgttgac</td><td> 60</td>

<td>attgcttaca</td><td>tcaaaatccc</td><td>gaacgctggc</td><td>cagatgcagc</td><td>cggtaaaggc</td><td>attcaaaatc</td><td> 120</td>

<td>cacaacaaaa</td><td>tctgggttat</td><td>cccggaacgt</td><td>gataccttta</td><td>ctaacccgga</td><td>agaaggtgac</td><td>ISO</td>

<td>ctgaacccgc</td><td>caccggaagc</td><td>gaaacaggtg</td><td>ccggtatctt</td><td>actatgactc</td><td>caactacctg</td><td> 240</td>

<td>tctaccgata</td><td>acgaaaagga</td><td>caactacctg</td><td>aaaggtgtta</td><td>ctaaactgtt</td><td>cgagcgtatt</td><td> 300</td>

<td>tactccaccg</td><td>acctgggccg</td><td>tatgctgctg</td><td>actageatcg</td><td>ttcgcggtat</td><td>cccgttctgg</td><td> 360</td>

<td>ggcggttcta</td><td>ccatcgatac</td><td>cgaactgaaa</td><td>gtaatcgaca</td><td>ctaactgcat</td><td>caacgttatt</td><td> 4 20</td>

<td>cagccggacg</td><td>gttactatcg</td><td>ttccgaagaa</td><td>ctgaacctgg</td><td>tgatcatcgg</td><td>cccgtctgct</td><td> 480</td>

<td>gatatcatcc</td><td>agttcgagtg</td><td>taagagcttt</td><td>ggtcacgaag</td><td>ttctgaacct</td><td>cacccgtaac</td><td> 540</td>

<td>ggctacggtt</td><td>ccactdagta</td><td>catccgtttc</td><td>tctccggact</td><td>tcaccttcgg</td><td>ttttgaagaa</td><td> 600</td>

<td>tccctggaag</td><td>tagacacgaa</td><td>cccactgctg</td><td>ggęgctggta</td><td>aattcgcaac</td><td>tgatcctgcg</td><td> 660</td>

<td>gttaccctgg</td><td>ctcacgaact</td><td>gattcatgca</td><td>ggccaccgcc</td><td>tgtacggtat</td><td>egccatcaat</td><td> 720</td>

<td>ccgaaccgtg</td><td>tcttcaaagt</td><td>taacaccaac</td><td>gcgtattacg</td><td>agatgtccgg</td><td>tctggaagtt</td><td> 790</td>

<td>agcttcgaag</td><td>aactgcgtac</td><td>ttttggcggt</td><td>cacgacgcta</td><td>aattcatcga</td><td>ctctctgcaa</td><td> 840</td>

<td>gaaaacgagt</td><td>tccgtctgta</td><td>ctactataac</td><td>aagttcaaag</td><td>atatcgcatc</td><td>caccctgaac</td><td> 900</td>

<td>aaagcgaaat</td><td>ccatcgtggg</td><td>taecactgct</td><td>tctctccagt</td><td>acatgaagaa</td><td>cgtttttaaa</td><td> 9 60</td>

<td>gaaaaatacc</td><td>tgctcagcga</td><td>agacacctcc</td><td>ggcaaattct</td><td>ctgtagacaa</td><td>gttgaaattc</td><td> 1020</td>

<td>gataaacttt</td><td>acaaaatgct</td><td>gactgaaatt</td><td>Latin cgaag</td><td>acaacttcgt</td><td>taagttcttt</td><td> 1080</td>

<td>aaagttctga</td><td>accgcaaaac</td><td>ctatctgaac</td><td>ttcgacaagg</td><td>cagtattcaa</td><td>aatcaacatc</td><td> 1140</td>

148

<td>gtgccgaaag</td><td>ctaactacac</td><td>tatctacgat</td><td>ggtttcaacc</td><td>tgcgtaacac</td><td>caacctggct</td><td> 1200</td>

<td>gctaatttta</td><td>acggccagaa</td><td>cacggaaatc</td><td>aacaacatga</td><td>acttcacaaa</td><td>acCgaaaaac</td><td> 1260</td>

<td>ttcactggtc</td><td>tgttcgagtt</td><td>ttacaagctg</td><td>ctgtgegtcg</td><td>aCggcatCat</td><td>tacctccaaa</td><td> 1320</td>

<td>actaaatctc</td><td>tgatcgaagg</td><td>tcgttacggt</td><td>ggtttcatga</td><td>cctctgaaaa</td><td>atctcagacc</td><td> 1380</td>

<td>ccgctggtta</td><td>ccCtgttcaa</td><td>aaacgctatc</td><td>atcaaaaacg</td><td>cttacaaaaa</td><td>aggtgaagcg</td><td> 144 0</td>

<td>ctagcgggtg</td><td>gtggtggttc</td><td>tggtggtggt</td><td>ggttctggtg</td><td>gtggtggttc</td><td>tgcactagtg</td><td> 1500</td>

<td>CTGcagtgta</td><td>tcaaggttaa</td><td>caactgggat</td><td>ttattcttca</td><td>gcccgagtga</td><td>agacaacttc</td><td> 1560</td>

<td>accaacgacc</td><td>tgaacaaagg</td><td>tgaagaaatc</td><td>acctcagata</td><td>ctaacatcga</td><td>agcagccgaa</td><td> 1620</td>

<td>gaaaacatct</td><td>cgctggacct</td><td>gatccagcag</td><td>tactacctga</td><td>cctttaattt</td><td>cgacaacgag</td><td> 1680</td>

<td>ccggaaaaca</td><td>tttctatcga</td><td>aaacctgagc</td><td>tctgatatca</td><td>tcggccagct</td><td>ggaactgatg</td><td> 1740</td>

<td>ccgaacatcg</td><td>aacgtttccc</td><td>aaacggtaaa</td><td>aagtacgagc</td><td>tggacaaata</td><td>taccatgttc</td><td> 1800</td>

<td>cactacctgc</td><td>gcgcgcagga</td><td>atttgaacac</td><td>ggcaaatccc</td><td>gtatcgcact</td><td>gactaactcc</td><td> 1860</td>

<td>gttaacgaag</td><td>ctctgctcaa</td><td>cccgtcccgt</td><td>gtatacacct</td><td>tcttctctag</td><td>cgactacgtg</td><td> 1920</td>

<td>aaaaaggtca</td><td>acaaagcgac</td><td>tgaagctgca</td><td>atgttcttgg</td><td>gttgggttga</td><td>acagcttgtt</td><td>19BO</td>

<td>tatgatttta</td><td>ccgacgagac</td><td>gtccgaagta</td><td>tctactaccg</td><td>acaaaattgc</td><td>ggatatcact</td><td> 2040</td>

<td>atcatcatcc</td><td>cgtacatcgg</td><td>tccggctctg</td><td>aacattggca</td><td>acatgctgta</td><td>caaagacgac</td><td> 2100</td>

<td>ttcgttggcg</td><td>cactgatctt</td><td>ctccggtgcg</td><td>gtgatcctgc</td><td>tggagttcat</td><td>cccggaaatc</td><td> 2160</td>

<td>gccatcccgg</td><td>tactgggcac</td><td>ctttgctctg</td><td>gtttettaca</td><td>ttgcaaacaa</td><td>ggttctgact</td><td> 2220</td>

<td>gtacaaacca</td><td>tcgacaacgc</td><td>gctgagcaaa</td><td>cgtaacgaaa</td><td>aatgggatga</td><td>agtttacaaa</td><td> 2280</td>

<td>tatatcgtga</td><td>ccaactggct</td><td>ggctaaggtt</td><td>aatacteaga</td><td>tcgacctcat</td><td>ccgcaaaaaa</td><td> 2340</td>

<td>atgaaagaag</td><td>cactggaaaa</td><td>ccaggcggaa</td><td>gctaecaagg</td><td>caatcattaa</td><td>ctaccagtac</td><td> 2400</td>

<td>aacaagtaca</td><td>your feet</td><td>aaaaaacaac</td><td>atcaacttca</td><td>acatcgacga</td><td>tctgtcctct</td><td> 2460</td>

<td>aaactgaacg</td><td>aatccatcaa</td><td>caaagctatg</td><td>atcaacatca</td><td>acaagttcat</td><td>gaaccagtgc</td><td> 2520</td>

<td>tctgtaagct</td><td>atctgatgaa</td><td>ctccatgatc</td><td>ccgtacggtg</td><td>ttaaacgtct</td><td>ggaggacttc</td><td> 2580</td>

<td>gatgcgtctc</td><td>tgaaagacgc</td><td>cctgctgaaa</td><td>tacatttacg</td><td>acaaccgtgg</td><td>cactctgatc</td><td> 2640</td>

<td>ggtcaggttg</td><td>atcgtctgaa</td><td>ggacaaagtg</td><td>aacaatacct</td><td>tatcgaccga</td><td>catccctctt</td><td> 2700</td>

<td>cagctcagta</td><td>aatatgtcga</td><td>taaccaacgc</td><td>cttttgtcca</td><td>ctctagacta</td><td>g</td><td> 2751</td>

&lt;210&gt; 24 &lt;211&gt; 916 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

149 &lt;223&gt; Synthetic &lt;400&gt; 24

150

Gly Ser Met Glu Phe Val Asn Lys Gin Phe Asn Tyr Lys Asp Pro Val 15 10 15

Asn Gly Val Asp Ile Ala Tyr Ile Lys 20 25

Ile Pro Asn Ala Gly Gin Met 30

Gin Pro

Glu Arg

Pro Glu 65

Thr cheese

Val Lys 35

Asp Thr

Ala Lys

Asp Asn

Ala phe

Phe Thr

Gin Val 70

Glu Lys 85

The Glu faction

Arg Background

100

Tyr Ser

How many Val

Arg Gly 115

How much Pro

Leu Lys

130

With Val

Asp Thr

Cheese Tyr 145

Arg Ser

Glu Glu

150

With Asp

How many Gin

The Glu 165 faction

Leu Thr

Arg Asn

180

Gly Tyr

Asp Phe

Thr phe 195

Gly Phe

Leu Leu

210

Gly Ala

Gly Lys

Lys Ile 40

Asn Pro 55

Pro Val

Asp Asn

Thr Asp

Phe Trp 120

Asn Cys 135

Leu Asn

Cys Lys

Gly Ser

Glu Glu 200

Phe Ala 215

His Asn

Glu Glu

Cheese Tyr

Tire of Leu 90

Leu Gly

105

Gly Gly

How many Asn

Leu Val

Phe cheese

170

Thr Gin 185

Leu cheese

Thr Asp

Lys Ile

Gly Asp 60

Tyr Asp 75

Lys Gly

Trp Val 45

Leu Asn

Thr cheese

Val Thr

How much Pro

Pro Pro

Tyr Leu 80

Lily Leu 95

Arg Met

Thr cheese

With Val

140

How many Ile 155

Gly His

Tyr Ile

Glu Val

Pro Ala

220

Leu Leu

110

Thr Sar

How many Asp 125

Thr Glu

Gin Pro

Asp Gly

Gly Pro

Glu Val

Arg Phe 190

Asp Thr 205

Val Thr

Ala cheese

160

Leu Asa 175

Pro cheese

Asn Pro

Leu Ala

His Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn

151

Thr Asn Ala Tyr Tyr Glu Met Ser

250 255

225

235

240

Pro Asn Arg Vai Phe Lys Val Asn.

245

Gly Leu Glu Val Cheese Phe Glu Glu

260

Leu Arg Thr Phe Gly Gly His Asp 265 270

Ala Lys Phe Ile Asp Ser Leu Gin

275 280

Glu Asn Glu Phe Arg Leu Tyr Tyr 235

Tyr Asn Lys Phe Lys Asp ile Ala

290 295

Thr Leu Asn Lys Ala Lys Ser 300

Ile Val Gly Thr Thr Ala Ser Leu 305 310

Gin Tyr Met Lys Asn Val Phe Lys

315 320

Glu Lys Tyr Leu

Leu Ser Glu Asp Thr Ser Gly Lys 325 330

Phe Cheese Val Asp 335

Lys Leu Lys Phe Asp Lys Leu Tyr

0

Lys Met Leu. Thr Glu Ile Tyr Thr 345 350

Glu Asp Asn Phe Val LyS Phe Phe

355 360

Lys Val Leu Asn Arg Lys Thr Tyr 365

Leu Asn Phe Asp Lys Ala Val Phe

370 375

Lys ile Asn ile Val Pro Lys Val

380

Asn Tyr Thr Ile Tyr Asp Gly Phe 385 390

Asn Leu Arg Asn Thr Asn Leu Ala

395 400

Ala Asn Phe Asn Gly Gin Asn Thr

405

Glu Background Asn Asn Met Asn Phe Thr

410 415

Lys Leu Lys Asn Phe Thr Gly Leu 420

Phe Glu Phe Tyr Lys Leu Leu Cys 425 430

Val Asp Gly Ile Ile Thr Ser Lys

435 440

Thr Lys Ser Leu Ile Glu Gly Arg 445

Leu 465

Tyr

<td>Gly 450</td><td>Gly</td><td>Phe</td><td>Underworld</td><td>Thr</td><td>Cheese 455</td><td>Glu</td><td>List</td><td>Cheese</td><td>Gin</td><td>Thr 460</td><td>Pro</td><td>Leu</td><td>Val</td><td>Thr</td>

<td>Phe</td><td>List</td><td>Asn</td><td>Ala</td><td>how much</td><td>How much</td><td>List</td><td>Asn</td><td>Ala</td><td>Tyr</td><td>List</td><td>List</td><td>Gly</td><td>Glu</td><td>Ala</td>

<td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

152

Gly Gly Ser Gly Gly Gly Gly

490 495

<td>Leu</td><td>Ala</td><td>Gly</td><td colspan="2">Gly Gly 485</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Gly</td>

<td>Cheese</td><td>Ala</td><td>Leu</td><td>Val 500</td><td>Leu</td><td>Gin</td><td>Cys</td><td>How much</td><td>Lys S05</td>

<td>Phe</td><td>Cheese</td><td>Pro SIS</td><td>Cheese</td><td>Glu</td><td>Asp</td><td>Asn</td><td>Phe 520</td><td>Thr</td>

Val Asn Asn Trp Asp Leu Phe 510

Asn Asp Leu Asn Lys Gly Glu 525

With Glu

530

Thr cheese

Asp Thr

Leu Asp 545

Leu Ile

Gin Gin

550

Pro Glu

Leu Glu

Glu Leu

Glu His

610

Leu Leu 625

Light Light

With Asn

Leu With 580

Asp Lys 595

Gly Lys

Asn Pro

Val Asn

With cheese 565

Pro Asn

Tyr Thr

Arg cheese

Arg cheese

630

Lys Ala 645

Glu Gin

Leu Val

660

Tire Asp

Thr Asp

Lys Ile 675

Ala Asp

Ala Leu

690

With Asn

Gly Asn

Leu Ile 705

Phe cheese

Gly Ala

710

Asn With 535

Tire Tire

Glu Asn

How much Glu

Met Phe

600

How Much Ala 615

Val Tyr

Thr Glu

Phe Thr

How Much Thr

680

With Leu 695

With Val

Glu Ala

Leu Thr

Leu Ser

570

Arg Phe 585

His Tyr

Leu Thr

Thr Phe

Do not start

650

Asp Glu 6 65

How many How many

Tyr Lys

Leu Leu

Ala Glu

540

Phe Asn 555

Asp cheese

Pro Asn

Leu Arg

Asn Ser

620

Phe Ser 635

Met Phe

Thr Ser

How much Pro

Asp Asp

700

Glu Phe 715

Glu Asn

Phe Asp

How many How many

Gly Lys 590

Ala Gin 605

Val Asn

Asp cheese

Leu Gly

Glu Val 670

Tire, House 685

Phe Val

How much Pro

How much Cheese

Asn Glu

0

Gly Gin 575

Light Tyr

Glu Phe

Glu Ala

Tire Val

640

Trp Val 655

Thr cheese

Gly Pro

Gly Ala

With Glu

720

153

Leu Val Ser Tyr Island Ala Asn

730 735

<td>Ala</td><td>How much</td><td>Pro</td><td>Val</td><td>Wow 725</td><td>Gly</td><td>Thr</td><td>Phe</td>

<td>List</td><td>Val</td><td>Leu</td><td>Thr 740</td><td>Val</td><td>Gin</td><td>Thr</td><td>How much</td>

<td>Glu</td><td>List</td><td>Trp 755</td><td>Asp</td><td>Glu</td><td>Val</td><td>Tyr</td><td>Lys 760</td>

<td>List</td><td>Val 770</td><td>Asn</td><td>Thr</td><td>Gin</td><td>How much</td><td>Asp 775</td><td>Leu</td>

<td>Leu 785</td><td>Glu</td><td>Asn</td><td>Gin</td><td>Ala</td><td>Glu 790</td><td>Ala</td><td>Thr</td>

<td>Asn</td><td>Gin</td><td>Tyr</td><td>Thr</td><td>Glu 805</td><td>Glu</td><td>Glu</td><td>List</td>

<td>Asp</td><td>Leu</td><td>Cheese</td><td>Cheese 820</td><td>List</td><td>Leu</td><td>Asn</td><td>Glu</td>

<td>How much</td><td>Asn</td><td>Lys 835</td><td>Phe</td><td>Wow</td><td>Asn</td><td>Gin</td><td>Cys 840</td>

<td>Underworld</td><td>How much 850</td><td>Pro</td><td>Tyr</td><td>Gly</td><td>Val</td><td>Lys 855</td><td>Arg</td>

<td>Lys 865</td><td>Aap</td><td>Ala</td><td>Leu</td><td>Leu</td><td>Lys 870</td><td>Tyr</td><td>How much</td>

<td>Gly</td><td>Gin</td><td>Val</td><td>Asp</td><td>Arg 885</td><td>Leu</td><td>List</td><td>Asp</td>

<td>Asp</td><td>How much</td><td>Pro</td><td>Phe</td><td>Gin</td><td>Wow</td><td>Cheese</td><td>List</td>

900

Asn Ala Leu Ser Lys Arg Asn 750

Ile Val Thr Asn Trp Leu Ala

765

Arg Lys Lys Met Lys Glu Ala 780

Ala With Asn Tyr Gin Tyr

795 800

Asn Ile Asn Phe Asn Ile Asp 810 815

Ile Asn Lys Ala Met Ile Asn

830

Val Ser Tyr Leu Met Asn Ser

845

Glu Asp Phe Asp Ala Ser Leu 860

Asp Asn Arg Gly Thr Leu Ile

875 880

Val Asn Asn Thr Leu Ser Thr

890 895

Val Asp Asn Gin Arg Leu Leu 910

Ser Thr Leu Asp 915 &lt;210&gt; 25 &lt;211&gt; 2709 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; synthetic

154 &lt;400&gt; 25 ggatccatgg agttcgttaa caaacagttc aactataaag acccagttaa cggtgttgac 60 attgcttaca tcaaaaŁccc gaacgctggc cagatgcagc cggtaaaggc attcaaaatc 120 cacaacaaaa tctgggttat cccggaacgt gataccttta ctaacccgga agaaggtgac 180 ctgaacccgc caccggaagc gaaacaggtg ccggtatctt actatgactc cacctacctg 240 tctaccgata acgaaaagga caactacctg aaaggtgtta ctaaactgtt cgagcgtatt 300 tactccaccg acctgggccg tatgctgctg actagcatcg ttcgcggtat cccgttctgg 360 ggcggttcta ccatcgatac cgaactgaaa gtaatcgaca ctaactgcat caaagttatt 420 cagccggacg gttcctatcg ttccgaagaa ctgaacctgg tgatcatcgg cccgtctgct 460 gatatcatcc agttcgagtg taagagcttt ggtcacgaag ttctgaacct cacccgtaac S4O ggctacggtt ccactcagta catccgtttc tctccggact tcaccttcgg ttttgaagaa 600 Łccctggaag tagacacgaa cccactgctg ggcgctggta aattcgcaac tgatcctgcg 660 gttaccctgg ctcacgaact gattcatgca ggccaccgcc tgtacggtat cgccatcaat 720 ccgaaccgtg tcttcaaagt taacaccaac gcgtattacg agatgtccgg tctggaagtt 780 agcttcgaag aactgcgtac ttttggcggt cacgacgcta aattcatcga ctctctgcaa 840 gaaaacgagt tccgtccgta ctactataac aagttcaaag atatcgcatc caccctgaac 900 aaagcgaaat ccatcgtggg taccactgct tctctccagt acatgaagaa cgtttttaaa 960 gaaaaatacc tgctcagcga agacacctcc ggcaaattct ctgtagacaa gttgaaattc 1020 gataaacttt acaaaatgct gactgaaatt tacaccgaag acaacttcgt taagttcttt 1080 aaagttctga accgcaaaac ctatctgaac ttcgacaagg cagtattcaa aatcaacatc 1140 gtgccgaaag ctaactacac tatctacgat ggtttcaacc tgcgtaacac caacctggct 1200 gctaatttta acggccagaa cacggaaatc aacaacatga acttcacaaa actgaaaaac 1260 ttcactggtc tgttcgagtt ttacaagctg ctgtgcgtcg acggcatcat tacctccaaa 1320 actaaatctc tgatagaagg tagatttggc ggtttcacgg gcgcacgcaa atcagcgcgt 1380 aaacgcgggggggggggggggggggggggggggg^cggtagcg cactagtgct gcagtgtatc aaggttaaca actgggattt attcttcagc 1500 ccgagtgaag acaacttcac caacgacctg aacaaaggtg aagaaatcac ctcagatact 1560 aacatcgaag cagccgaaga aaacatctcg ctggacctga tccagcagta ctacctgacc 1620 tttaatttcg acaacgagcc ggaaaacatt tctatcgaaa acctgagctc tgatatcatc 1660 ggccagctgg aactgatgcc gaacatcgaa cgtttcccaa acggtaaaaa gtacgagctg 1740 gacaaatata ccatgttcca ctacctgcgc gcgcaggaat ttgaacacgg caaatcccgt 1800

155 atcgcactga ctaactccgt taacgaagct ctgctcaacc cgtcccgtgt atacaccttc 1860 ttctctagcg actacgtgaa aaaggtcaac aaagcgactg aagctgcaat gttcttgggt 1920 tgggttgaac agcttgctta tgattttacc gacgagacgt ccgaagtatc tactaccgac 1980 aaaattgcgg atatcactat catcatcccg tacatcggtc cggctctgaa cattggcaac 2040 atgctgtaca aagaagactt cgttggcgca ctgatcttct ccggtgcggt gatcctgctg 2100 gagttcatcc cggaaatcgc catcccggta ctgggcacct ttgctctggt ttcttacatt 2160 gcaaacaagg ttctgactgt acaaaccatc gacaacgcgc tgagcaaacg taacgaaaaa 2220 tgggatgaag tttacaaata tatcgtgacc aactggctgg ctaaggttaa tactcagatc 2280 gacctcatcc gcaaaaaaat gaaagaagca ctggaaaacc aggcggaagc taccaaggca 2340 atcattaact accagtacaa ccagtacacc gaggaagaaa aaaacaacat caacttcaac 2400 atcgacgatc tgtcctctaa actgaacgaa tccatcaaca aagctatgat caacatcaac 2460 aagttcctga accagtgctc tgtaagctat ctgatgaact ccatgatccc gtacggtgtt 2520 aaacgtctgg aggacttcga tgcgtctctg aaagacgccc tgctgaaata catttacgac 2580 aaccgtggca ctctgatcgg tcaggttgat cgtctgaagg acaaagtgaa caatacctta 2640 tcgaccgaca tcccttttca gctcagtaaa tatgtcgata accaacgcct tttgtccact 2700 atagactag 2709 &lt;210&gt; 26 &lt;211&gt; 902 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 26

156

Gly Cheese Met Glu Phe 1 5

Asn Gly Vał Asp Ile 20

Gin Pro Val Lys Ala 35

Glu Arg Asp Thr Phe 50

Val Asn Lys Gin Phe Asn 10

Ala Tyr Ile Łys Ile Pro 25

Phe Lys Ile His Asn Lys 40

Thr Asn Pro Glu Glu Gly 55

Tyr Lys Asp Pro Val 15

Asn Ala Gly Gin Met 30

How many Trp Val Ile Pro 45

Asp Leu Asn Pro Pro 60

Pro Glu Ala Lys Gin Val

70

Pro Val

Ser Tyr Tyr Asp Ser Thr Tyr Leu 75 80

157 Thr Asp Asn Glu cheese 85

Lys Asp Asn Tyr Leu

Lys Gly Val Thr Lys 95

Phe Glu Arg Ile Tyr 100

Thr Asp Leu Gly cheese 105

Arg Met Leu Leu Thr 110

Ile Val Arg With Gly 115

Pro Phe Trp Gly Gly 120

Cheese Thr Ile Asp Thr 125

Leu Lys Val Ile Asp 130

Thr Asn Cys ile Asn 135

Gin Pro Asp 140 With Val

Tyr Arg Cheese Glu 145

Glu Leu Asn Leu Val 150

How Much Ile Gly Pro Ser 155

Gin Phe With Asp

165

Glu Cys Lys Ser Phe

170

Gly His Glu Val Leu

175

Leu Thr Arg Asn Gly 180

Tyr Gly Ser Thr Gin

185

Tyr Ile Arg Phe Ser 190

Asp Phe Thr Phe Gly 195

The Glu Glu Ser Leu 200

Glu Val Asp Thr Asn 205

Leu Leu Gly Ala Gly 210

Lys Phe Ala Thr Asp 215

Pro Ala Val Thr Leu

220

His Glu Leu with His 225

Ala Gly His Arg Leu 230

23S With Ala With Tyr Gly

Pro Asn Arg Val Phe

245

Lys Val Asn Thr Asn

250

Ala Tyr Tyr Glu Met

255

Gly Leu Glu Val Ser 260

Phe Glu Glu Leu Arg 265

Thr Phe Gly Gly His 270

Ala Lys Phe Ile Asp 275

Cheese Leu Gin Glu Asn

280

Glu Phe Arg Leu Tyr 285

Tyr Asn Lys Phe Lys 290

Asp Ile Ala Ser Thr 295

Leu Asn Lys Ala Lys

300

Ile Val Gly Thr Thr 305

Ala Ser Leu Gin Tyr 310

With List Asn Val Phe 315

Leu

Cheese

Glu

Gly

Ala 160

Asn

Pro

Pro

Ala

Asn 240

Cheese

Asp

Tyr

Cheese

Lys 320

158

Glu Lys Tyr Leu Leu Ser 325

Lys Leu Lys Phe Asp Lys 340

Glu Asp Asn Phe Val Lys 3 55

Leu Asn Phe Asp Lys Ala 370

Glu Asp

Leu Tyr

Phe Phe

60

Val Phe 375

Asn Tyr Thr 385

Ile Tyr Asp

390

Thr Ser Gly Lys Phe Ser Val Asp

330 335

Lys Met Leu Thr Glu Ile Tyr Thr 345 350

Lys Val Leu Asn Arg Lys Thr Tyr 365

Lys ile Asn ile Val Pro Lys Val

380

Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala

395 400

Ala Asn Phe

Lys Leu Lys

<td>Asn</td><td>Gly 405</td><td>Gin Asn</td><td>Thr</td><td>Glu</td><td>How much 410</td><td>Asn Asn</td><td>Underworld</td><td>Asn Phe 415</td><td>Thr</td>

<td>Asn 420</td><td>Phe</td><td>Thr Gly</td><td>Leu</td><td>Phe 425</td><td>Glu</td><td>Phe Tyr</td><td>List</td><td>Leu Leu 430</td><td>Cys</td>

Val Asp Gly Ile

435

Ile Thr Ser Lys Thr Lys Ser Leu Ile

440 445

Glu Gly Arg

Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala Arg Lys Arg Lys Asn

450 455 460

Gin Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 465 470 475 480

Gly Gly Ser Ala Leu Val Leu Gin Cys Ile Lys Val Asn Asn Trp Asp 485 490 495

Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn. Bald

500 SOS 510

Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn 515 520 52S

Ile Ser Leu Asp Leu Ile Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp

530 535 540

Asn Glu Pro Glu Asn Ile Cheese Ile Glu Asn Leu Cheese Cheese Asp Ile Ile 545 550 5SS 560

159

Glu Arg Phe Pro Asn Gly Lys

570 575

<td>Gly</td><td>Gin</td><td>Leu</td><td>Glu</td><td>Leu 565</td><td>Underworld</td><td>Pro</td><td>Asn</td>

<td>List</td><td>Tyr</td><td>Glu</td><td>Leu 580</td><td>Asp</td><td>List</td><td>Tyr</td><td>Thr</td>

<td>Glu</td><td>Phe</td><td>Glu 595</td><td>His</td><td>Gly</td><td>List</td><td>Cheese</td><td>Arg 600</td>

<td>Glu</td><td>Ala 610</td><td>Leu</td><td>Leu</td><td>Asn</td><td>Pro</td><td>Cheese 615</td><td>Arg</td>

<td>Tyr 625</td><td>Val</td><td>List</td><td>List</td><td>Val</td><td>Asn 630</td><td>List</td><td>Ala</td>

<td>Trp</td><td>val</td><td>Glu</td><td>Gin</td><td>Leu 645</td><td>Val</td><td>Tyr</td><td>Asp</td>

<td>cheese</td><td>Thr</td><td>Thr</td><td>Asp 660</td><td>List</td><td>How much</td><td>Ala</td><td>Asp</td>

<td>Gly</td><td>Pro</td><td>Ala 675</td><td>Leu</td><td>Asn</td><td>How much</td><td>Gly</td><td>Asn 680</td>

<td>Gly</td><td>Ala 690</td><td>Leu</td><td>How much</td><td>Phe</td><td>Cheese</td><td>Gly 695</td><td>Ala</td>

<td>Glu 705</td><td>How much</td><td>Aid</td><td>How much</td><td>Pro</td><td>Val 710</td><td>Leu</td><td>Gly</td>

<td>Ala</td><td>Asn</td><td>List</td><td>val</td><td>Leu 7Ξ5</td><td>Thr</td><td>Val</td><td>Gin</td>

<td>Arg</td><td>Asn</td><td>Glu</td><td>Lys 740</td><td>Trp</td><td>Asp</td><td>Glu</td><td>Val</td>

<td>Leu</td><td>Ala</td><td>Lys 755</td><td>Val</td><td>Asn</td><td>Thr</td><td>Gin</td><td>How much 760</td>

<td>Glu</td><td>Ala 770</td><td>Leu</td><td>Glu</td><td>Asn</td><td>Gin</td><td>Ala 775</td><td>Glu</td>

<td>Gin 7Θ5</td><td>Tyr</td><td>Asn</td><td>Gin</td><td>Tyr</td><td>Thr 790</td><td>Glu</td><td>Glu</td>

Phe His Tyr Leu Arg Ala Gin 590

Ala Leu Thr Asn Ser Val Asn 605

Tyr Thr Phe Phe Ser Ser Asp 620

Glu Ala Ala Met phe Leu Gly

635 640

Thr Asp Glu Thr Ser Glu val

650 655

With Thr With Pro With Tyr 670

Leu Tyr Lys Asp Asp Phe Val 685

How many Leu Leu Glu Phe Ile Pro 700

Phe Ala Leu Val Ser Tyr Ile

715 720

Ile Asp Asn Ala Leu Ser Lys 730 735

Lys Tyr Ile Val Thr Asn Trp

750

Leu Ile Arg Lys Lys Met Lys 765

Thr Lys Ala Ile Ile Asn Tyr 780

Lys Asn Asn Ile Asn Phe Asn

795 800

Ile Asp Asp Leu Cheese Lys Leu Asn Glu Cheese Ile Asn Lys Ala Met

160

Underworld

805

810

Θ15

Asn

How much

Asn 820

List

Phe

Leu

Asn

Gin

825

Cys

Cheese

Val

Cheese

Tyr

830

Wow

Asn

Cheese

Underworld

835

How much

Pro

Gly

Val

840

List

Arg

Leu

Glu

Asp

845

Phe

Asp

Ala

Cheese

Leu

850

List

Asp

Ala

Leu

Leu

855

List

Tyr

How much

Asp 860

Asn.

Arg

Gly

Thr

Leu 065

How much

Gly

Gin

Val

Asp 870

Arg

Leu

List

Asp

List

875

Val

Asn

Asn

Thr

Leu

880

Cheese

Thr

Asp

How much

Pro 885

Phe

Gin

Leu

Cheese

Lys 890

Tyr

Val

Asp

Asn

Gin

895

Arg

Leu Leu Ser Thr Leu Asp

900 &lt;210&gt; 27 &lt;211&gt; 2691 &lt;212&gt; DNA &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 27

161

<td>ggatccatgg</td><td>agttcgttaa</td><td>caaacagttc</td><td>aactataaag</td><td>acccagttaa</td><td>cggtgttgac</td><td> 60</td>

<td>attgcttaca</td><td>tcaaaatccc</td><td>gaacgctggc</td><td>cagatgcagc</td><td>cggtaaaggc</td><td>attcaaaatc</td><td> 120</td>

<td>cacaacaaaa</td><td>tctgggttat</td><td>cccggaacgt</td><td>gataccttta</td><td>ctaacccgga</td><td>agaaggtgac</td><td>ISO</td>

<td>ctgaacccgc</td><td>caccggaagc</td><td>gaaacaggtg</td><td>ccggtatctt</td><td>actatgactc</td><td>cacctacctg</td><td> 240</td>

<td>tctaacgata</td><td>acgaaaagga</td><td>caaccacctg</td><td>aaaggtgtta</td><td>ctaaactgtt</td><td>cgagcgtatt</td><td> 300</td>

<td>tactccaccg</td><td>acctgggccg</td><td>tatgctgctg</td><td>actagcatcg</td><td>ttcgcggtat</td><td>cccgttctgg</td><td> 360</td>

<td>ggcggttcta</td><td>ccatcgatac</td><td>cgaactgaaa</td><td>gtaatcgaca</td><td>ctaactgcat</td><td>caacgttatt</td><td> 420</td>

<td>cagccggacg</td><td>gttectatcg</td><td>tEccgaagaa</td><td>ctgaacctgg</td><td>tgatcatcgg</td><td>cccgtctgct</td><td> 480</td>

<td>gatatcatcc</td><td>agttcgagtg</td><td>taagagcttt</td><td>ggtcacgaag</td><td>ttctgaacct</td><td>cacccgtaac</td><td> 540</td>

<td>ggctacggtt</td><td>ccactcagta</td><td>catccgtCtc</td><td>Łctccggact</td><td>tcaccttcgg</td><td>ttttgaagaa</td><td> 600</td>

<td>tccctggaag</td><td>tagacacgaa</td><td>cccactgctg</td><td>ggcgatggta</td><td>aattcgcaac</td><td>tgatcctgcg</td><td> 660</td>

<td>gttaccctgg</td><td>ctcacgaact</td><td>gatEcatgca</td><td>ggccaccgcc</td><td>tgtacggtat</td><td>cgccatcaat</td><td> 720</td>

<td>ccgaaccgtg</td><td>tcttcaaagt</td><td>taacaccaac</td><td>gcgtattacg</td><td>agatgtccgg</td><td>tctggaagtt</td><td> 780</td>

162

<td>agcttcgaag</td><td>aactgcgtac</td><td>ttttggcggt</td><td>cacgacgcta</td><td>aattcatcga</td><td>ctctctgcaa</td><td> 640</td>

<td>gaaaacgagt</td><td>tccgtctgta</td><td>ctactataac</td><td>aagttcaaag</td><td>atatcgcatc</td><td>caccctgaac</td><td> 900</td>

<td>aaagcgaaat</td><td>ccatcgtggg</td><td>taccactgct</td><td>tctctccagt</td><td>acatgaagaa</td><td>cgtttttaaa</td><td> 960</td>

<td>gaaaaataca</td><td>tgctcagcga</td><td>agacacctcc</td><td>ggcaaattct</td><td>ctgtagacaa</td><td>gttgaaattc</td><td> 1020</td>

<td>gataaacttt</td><td>acaaaatgct</td><td>gactgaaatt</td><td>Latin cgaag</td><td>acaacttcgt</td><td>taagttcttt</td><td>10S0</td>

<td>aaagttctga</td><td>accgcaaaac</td><td>ctatctgaac</td><td>ttcgacaagg</td><td>cagtattcaa</td><td>aatcaacatc</td><td> 1140</td>

<td>gtgccgaaag</td><td>ttaactacac</td><td>tatctacgat</td><td>ggtttcaacc</td><td>tgcgeaacac</td><td>caacctggct</td><td> 1200</td>

<td>gctaatttta</td><td>acggccagaa</td><td>cacggaaatc</td><td>aacaacatga</td><td>acttcacaaa</td><td>actgaaaaac</td><td> 1260</td>

<td>ttcactggtc</td><td>tgttcgagtt</td><td>ttacaagctg</td><td>ctgtgcgtcg</td><td>acggcatcat</td><td>tacctccaaa</td><td> 1320</td>

<td>actaaatctc</td><td>tgatagaagg</td><td>tagatttggc</td><td>ggtttcacgg</td><td>gcgcacgcaa</td><td>atcagcggeg</td><td>13Θ0</td>

<td>ctagcgggcg</td><td>gtggcggtag</td><td>cggcggtggc</td><td>ggtagcggcg</td><td>gtggcggtag</td><td>cgcactagtg</td><td> 1440</td>

<td>CTGcagtgta</td><td>tcaaggttaa</td><td>caactgggat</td><td>ttattcttca</td><td>gcccgagtga</td><td>agacaacttc</td><td> 150 0</td>

<td>accaacgacc</td><td>tgaacaaagg</td><td>tgaagaaatc</td><td>acctcagata</td><td>ctaacatcga</td><td>agcagccgaa</td><td> 1560</td>

<td>gaaaacatct</td><td>cgctggacct</td><td>gatccagcag</td><td>tactacctga</td><td>cctttaattt</td><td>cgacaacgag</td><td> 1620</td>

<td>ccggaaaaca</td><td>tttctatcga</td><td>aaacctgagc</td><td>tctgatatca</td><td>tcggccagct</td><td>ggaactgatg</td><td> 1680</td>

<td>ccgaacatcg</td><td>aacgtttccc</td><td>aaacggtaaa</td><td>aagtacgagc</td><td>tggacaaata</td><td>taccatgttc</td><td> 1740</td>

<td>cactacctgc</td><td>gcgcgcagga</td><td>atttgaacac</td><td>ggcaaatccc</td><td>gtatcgcact</td><td>gactaactcc</td><td> 1300</td>

<td>gttaacgaag</td><td>ctctgctcaa</td><td>cccgtcccgt</td><td>gtatacacet</td><td>tcttctatag</td><td>cgactacgtg</td><td> 1860</td>

<td>aaaaaggtca</td><td>acaaagcgac</td><td>tgaagctgca</td><td>atgttcttgg</td><td>gttgggttga</td><td>acagcttgtt</td><td> 1920</td>

<td>tatgatttta</td><td>ccgacgagac</td><td>gtccgaagta</td><td>tctactaccg</td><td>acaaaattgc</td><td>ggatatcact</td><td> 1980</td>

<td>atcatcatcc</td><td>cgEacatcgg</td><td>tccggctatg</td><td>aacattggca</td><td>acatgctgta</td><td>caaagacgac</td><td> 2040</td>

<td>ttcgttggcg</td><td>cactgatctt</td><td>ctccggtgcg</td><td>gtgatcctgc</td><td>tggagttcat</td><td>cccggaaatc</td><td> 2100</td>

<td>gccatcccgg</td><td>tactgggcac</td><td>ctttgctctg</td><td>gtttcttaca</td><td>ttgcaaacaa</td><td>ggttctgact</td><td> 2160</td>

<td>gtacaaaCca</td><td>tcgacaacgc</td><td>gctgagcaaa</td><td>cgtaacgaaa</td><td>aatgggatga</td><td>agtttacaaa</td><td> 2220</td>

<td>tatatcgtga</td><td>ccaactggct</td><td>ggctaaggtt</td><td>aatactcaga</td><td>tcgacctcat</td><td>ccgcaaaaaa</td><td> 2280</td>

<td>atgaaagaag</td><td>cactggaaaa</td><td>ccaggcggaa</td><td>gctaccaagg</td><td>caatcattaa</td><td>ctaccagtac</td><td> 2340</td>

<td>aaccagtaca</td><td>your feet</td><td>3.aasa.3CdBC</td><td>atcaacttca</td><td>acatcgacga</td><td>tctgtcctct</td><td> 2400</td>

<td>aaactgaacg</td><td>aatccatcaa</td><td>caaagctatg</td><td>atcaacatca</td><td>acaagttcct</td><td>gaaccagtgc</td><td> 2460</td>

<td>tctgtaagct</td><td>atctgatgaa</td><td>ctccatgatc</td><td>ccgtacggtg</td><td>ttaaacgtct</td><td>ggaggacttc</td><td> 2520</td>

<td>gatgcgtctc</td><td>tgaaagacgc</td><td>cctgctgaaa</td><td>tacatttacg</td><td>acaaccgtgg</td><td>cactctgatc</td><td> 2580</td>

163 ggtcaggttg atcgtctgaa ggacaaagtg aacaatacct tatcgaccga catccctttt cagctcagta aatatgtcga taaccaacgc cttttgtcca ctctagacta g

2640

2691 &lt;210&gt; 28 &lt;211&gt; 896 &lt;212&gt; PRT &lt;213&gt; Dummy &lt;220&gt;

&lt;223&gt;

Synthetic &lt;400&gt; 28

164

Gly Ser Met Glu Phe Val Asn List 1 5

Phe Asn Tyr Lys Asp Pro Val 10 15

Asn Gly Val Asp ile Ala Tyr 20

Ile Pro Asn Ala Gly Gin Met

Gin Pro Val Lys Ala Phe Lys Ile 35 40

Asn Lys With Trp Val Pro 45

Glu Arg Asp Thr Phe Thr Asn Pro

55

Glu Gly Asp Leu Asn Pro Pro 60

Pro Glu Ala Lys Gin Val Pro Val 65 70

Tyr Tyr Asp Ser Thr Tyr Leu eo

Ser Thr Asp Asn Glu Lys Asp Asn

Leu Lys Gly Val Thr Lys Leu 90 95

Phe Glu Arg Ile Tyr Ser Thr Asp

100

Gly Arg Met Leu Leu Thr Ser 110

Pro Phe Trp with Ile Val Arg Gly

115 120

Gly Ser Thr Ile Asp Thr Glu 125

Leu Lys Val Ile Asp Thr Asn Cys. 130 '135

Asn Val Ile Gin Pro Asp Gly 140

Tyr Arg cheese Glu Glu Leu Asn cheese 145 150

Get Gly Pro Ser with Val

155 160

Gin Phe Glu Cys Lys 165 With Asp

Phe Gly His Glu Val Leu Asn 170 175

Leu Thr Arg Asn Gly Tyr Gly ser 180

Gin Tyr Ile Arg Phe Cheese Pro 190

165

<td>Asp</td><td>Phe</td><td>Thr 195</td><td>Phe</td><td>Gly</td><td>Phe</td><td>Glu</td><td>Glu 200</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Val</td><td>Asp 205</td><td>Thr</td><td>Asn</td><td>Pro</td>

<td>Leu</td><td>Leu 210</td><td>Gly</td><td>Ala</td><td>Gly</td><td>List</td><td>Phe 215</td><td>Ala</td><td>Thr</td><td>Asp</td><td>Pro</td><td>Ala 220</td><td>Val</td><td>Thr</td><td>Leu</td><td>Ala</td>

<td>His 225</td><td>Glu</td><td>Leu</td><td>How much</td><td>His</td><td>Ala 230</td><td>Gly</td><td>His</td><td>Arg</td><td>Leu</td><td>Tyr 235</td><td>Gly</td><td>How much</td><td>Ala</td><td>Background</td><td>Asn 240</td>

<td>Pro</td><td>Asn</td><td>Arg</td><td>Val</td><td>Phe 245</td><td>List</td><td>Val</td><td>Asn</td><td>Thr</td><td>Asn 250</td><td>Ala</td><td>Tyr</td><td>Tyr</td><td>Glu</td><td>Underworld 255</td><td>Cheese</td>

<td>Gly</td><td>Leu</td><td>Glu</td><td>Val 260</td><td>Cheese</td><td>Phe</td><td>Glu</td><td>Glu</td><td>Leu 265</td><td>Arg</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Gly 27 0</td><td>His</td><td>Asp</td>

<td>Ala</td><td>List</td><td>Phe 27S</td><td>How much</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Gin 280</td><td>Glu</td><td>Asn</td><td>Glu</td><td>Phe</td><td>Arg 285</td><td>Leu</td><td>Tyr</td><td>Tyr</td>

<td>Tyr</td><td>Asn 290</td><td>List</td><td>Phe</td><td>List</td><td>Asp</td><td>How much 295</td><td>Ala</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Asn 300</td><td>List</td><td>Ala</td><td>List</td><td>Cheese</td>