Method for preparing proteolytically treated polypeptides

Abstract

The invention relates to a method for preparing a polypeptide subjected to proteolytic treatment, belongs to the field of protein engineering, and particularly relates to a novel polypeptide with proteolytic activity and various applications of the polypeptide (and others) in screening and preparation methods. The present invention addresses the technical problem of efficiently converting a single-stranded precursor CNT into a real mature cleavage product, i.e., a double-stranded neurotoxin, by providing a protease having a specific sequence.

Description

[0001] This application is a divisional application of the application with the application number 202010161444.9, titled "Method for Preparing Protein Hydrolyzed Polypeptides", filed on November 21, 2012.

[0002] The present invention relates to novel polypeptides having proteolytic activity and various applications of such polypeptides in screening and preparation methods.

[0003] Clostridium botulinum and Clostridium tetani produce highly potent neurotoxins, namely, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT), respectively. These clostridial neurotoxins (CNT) specifically bind to neuronal cells and interfere with the release of neurotransmitters. Clostridium botulinum secretes seven differentially antigenic serotypes of botulinum neurotoxin (BoNT) named A to G. All serotypes, as well as the related tetanus neurotoxin (TeNT) secreted by Clostridium tetani, are Zn 2 + -endoproteases that effect the above blockade by cleaving proteins involved in the formation of the SNARE complex that controls cell membrane fusion. CNT causes the flaccid muscle paralysis seen in botulism and tetanus. In addition, CNT activity has also been shown to affect glandular secretion. These physiological effects of CNT on muscle and glandular activity are increasingly being used in various therapeutic and cosmetic applications. In 1989, the United States approved the use of botulinum neurotoxin serotype A (BoNT / A) in humans for the treatment of strabismus, blepharospasm, and other disorders. It is commercially available as a botulinum neurotoxin A protein preparation, for example, sold under the trade name BOTOX (Allergan) and the trade name DYSPORT (Ipsen Ltd.). For therapeutic applications, a complex containing the neurotoxin and other bacterial proteins is typically directly injected into the muscle to be treated. At physiological pH, the toxin is released from the protein complex (Eisele et al. 2011, Toxicon 57(4):555-65.) and subsequently exerts the desired pharmacological effect. An improved BoNT / A preparation without complex proteins is commercially available, sold under the trade name XEOMIN or Bocouture (Merz Pharma GmbH & Co. KGaA, Frankfurt, Germany). The effect of BoNT is only temporary, so repeated administration of BoNT is often required to maintain the therapeutic effect.

[0004] Initially, each CNT is synthesized as an inactive single-chain polypeptide. In the case of BoNT, the molecular weight of this neurotoxin polypeptide is approximately 150 kDa. The post-translational processing of this single-chain polypeptide involves limited proteolysis in the exposed regions called loops (see Table 1) and the formation of nearby disulfide bonds. The activated double-chain neurotoxin consists of two cleavage products generated by proteolysis of the single-chain precursor polypeptide: an N-terminal light chain of approximately 50 kDa and a heavy chain of approximately 100 kDa, which are linked by a disulfide bond. Structurally, CNT is composed of three domains, namely, a catalytic light chain, and a heavy chain containing a translocation domain (the N-terminal half) and a receptor-binding domain (the C-terminal half) (see Krieglstein 1990, Eur J Biochem 188:39; Krieglstein 1991, Eur J Biochem 202:41; Krieglstein 1994, J Protein Chem 13:49; Lacy et al., 1998, Nat. Struct. Biol. 5(10):898-902). Depending on the number of cleavage sites present on the single chain, between the amino acid residues forming the catalytic domain and those forming the translocation domain, endopeptidase activity can generate two large cleavage products (i.e., the light chain and the heavy chain), as well as characteristic short peptides representing the loop regions that bridge the single chain of the neurotoxin (which later form the light chain and the heavy chain) (see Table 1 below).

[0005] Purifying CNT from the fermentation solution is particularly difficult because it contains a mixture of unprocessed, partially processed, and fully processed polypeptides, all of which have very similar biochemical and physical properties. If the endoproteolytic activity hydrolyzes the peptide bond between the light chain and the loop, while the peptide bond between the loop and the N-terminus of the heavy chain remains intact, partially processed neurotoxins are usually generated. In addition, if the endoproteolytic activity releases the loop peptide from the heavy chain, while the peptide bond between the loop peptide and the C-terminus of the light chain has not been hydrolyzed, partially processed neurotoxins are also generated. Depending on the fermentation conditions and the type of neurotoxin, fully processed polypeptides without loop peptides can be severely contaminated with 5% - 99% partially processed or unprocessed polypeptides. And in some cases, the neurotoxin is mainly unprocessed and needs to be treated with endopeptidase to become biologically active before therapeutic application.

[0006] A variety of methods for treating Clostridium botulinum neurotoxin with heterologous proteases to reduce the amount of untreated or partially treated precursor proteins have been described in the prior art. The protease most widely used to activate Clostridium botulinum neurotoxin is trypsin, which can also usefully activate Clostridium botulinum neurotoxin serotypes B (BoNT / B) and E (BoNT / E) (DasGupta and Sugiyama 1972, Biochem. Biophys. Res. Commun. 48: 108-112; Kozaki et al., 1974, Infect. Immun. 10: 750-756), but it seems to produce secondary products (presumably by proteolysis of the C-terminus of the heavy chain subunit near BoNT / A), thus seemingly disrupting the binding of the toxin to its cell receptor (Shone et al., 1985, Eur. J. Biochem. 151: 75-82). In theory, an endogenous protease isolated from a natural host (e.g., Clostridium botulinum that produces BoNT / A) should be able to produce more specific cleavage products. Therefore, several attempts have been made to isolate from natural host cells the endogenous protease involved in the proteolytic activation of Clostridium botulinum neurotoxin. Dekleva and DasGupta (Dekleva and DasGupta, 1989, Biochem. Biophys. Res. Commun. 162: 767-772) purified from a Clostridium botulinum culture that produces BoNT / A a component capable of proteolytically cleaving BoNT / A into the heavy and light chain subunits. Subsequent studies by the same authors further characterized the endogenous protease isolated from Clostridium botulinum (Dekleva and DasGupta, 1990, J. Bact. 172: 2498-2503) and revealed a 62 kDa protein composed of a 15.5 kDa polypeptide and a 48 kDa polypeptide. However, extensive cleavage of CNT was observed after limited exposure to Dekleva and DasGupta's 62 kDa protein, indicating that this isolated protease may not be the unrecognized protease responsible for activating CNT during Clostridium botulinum cell culture and infection. In fact, other researchers have recently pointed out that clostripain, also known as clostridiopeptidase B (Mitchel and Harrington, 1968, JBC 243: 4683-4692) may be involved in the specific activation of CNT (Sebaihia et al., 2007, Genome Res. 17(7): 1082-1092; WO2009 / 014854).Interestingly, the structure and substrate specificity of the enzyme are reminiscent of those of the α-clostripain secreted by Clostridium histolyticum (Dargatz et al., 1993), and its homolog (with 74% amino acid identity) is present in Clostridium botulinum (CBO1920). Clostridium histolyticum α-clostripain is a cysteine endopeptidase with the most stringent specificity for arginyl bonds. It is synthesized as an inactive prepro-enzyme, which undergoes autocatalytic cleavage to generate polypeptides of 15.4 and 43 kDa, which are linked together to form a heterodimeric active enzyme (Dargatz et al., 1993). Both Clostridium histolyticum α-clostripain and the 62-kDa protease of Clostridium botulinum require a reducing agent and calcium for full activity and are sensitive to the same protease inhibitors. These data strongly suggest that the direct homolog of α-clostripain in Clostridium botulinum (CBO1920) is the endogenous protease responsible for proteolytic cleavage of the neurotoxin of Clostridium botulinum. The gene encoding clostripain (CPE0846) is also present in Clostridium perfringens, and it was found that this gene is positively regulated by the two-component system VirR / VirS (Shimizu et al., 2002b).

[0007] To date, there is still a lack of further conclusive experimental evidence, and there is still no protease in this field that can effectively convert the single-chain precursor CNT into a truly mature cleavage product (i.e., the double-chain neurotoxin). The present invention solves one or more of the above problems.

[0008] A method for reducing unprocessed and / or partially processed neurotoxin peptides and thereby improving the quality of neurotoxin preparations is highly needed but not yet available. Therefore, the potential technical problem of the present invention can be regarded as providing means and methods for improving the preparation of neurotoxin polypeptides by meeting the aforementioned needs. This technical problem is solved by the following claims and the embodiments described hereinafter.

[0009] Accordingly, one aspect of the present invention relates to a polypeptide having proteolytic activity, which comprises a polypeptide sequence having at least 50% sequence identity with SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide having proteolytic activity, which consists of a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. In another aspect, the present invention relates to a polypeptide having proteolytic activity, which consists of the polypeptide sequence shown in SEQ ID NO: 1.

[0010] As used herein, the term "polypeptide with proteolytic activity" refers to the catalytic function of the polypeptides of the present invention and means that the polypeptides of the present invention are capable of hydrolyzing peptide bonds. In one aspect, the "polypeptide with proteolytic activity" refers to a polypeptide capable of hydrolyzing a polypeptide comprising any one of the amino acid sequences selected from SEQ ID NOs: 4 to 25. As used herein, the term "polypeptide without proteolytic activity" refers to the catalytic function of the polypeptides of the present invention and means that the polypeptides of the present invention are unable to hydrolyze peptide bonds.

[0011] By testing the proteolytic activity of the polypeptide, one skilled in the art can determine the polypeptide defined according to the sequence described herein. A test or assay system for detecting proteolytic activity includes: contacting a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1 with a test substrate. The test substrate is typically a polypeptide known to be cleaved by the polypeptides of the present invention. Preferably, the test substrate is CNT, such as BoNT or a fragment thereof. The test substrate can be, for example, uncleaved / untreated BoNT, referred to herein as "scBoNT", and can be, for example, serotype A, B, C1, D, E, F, or G (e.g., "scBoNT / A", "scBoNT / B", etc.) or the test substrate can be tetanus neurotoxin. Alternatively, the test substrate can be a fragment of a Clostridium botulinum neurotoxin, the fragment comprising any one of the amino acid sequences selected from SEQ ID NOs: 4 to 25. The fragment can be a polypeptide having 50 or more amino acid residues, or a peptide having up to 49 amino acid residues. As used throughout this specification, the term "polypeptide" refers to a molecule having 50 or more amino acids, while the term "peptide" refers to a molecule having 2 to 49 amino acid residues. In one aspect, the test substrate is a soluble neurotoxin fragment designated LH N that comprises a light chain polypeptide, an exposed loop peptide region, and the N-terminal half of the heavy chain polypeptide (i.e., the translocation domain H N ). In another aspect, the test substrate is, or comprises, a peptide selected from any one of SEQ ID NOs: 4 to 25 (see Table 1). And in another aspect, the test substrate is a chimeric neurotoxin that comprises amino acid residues derived from two or more serotypes.

[0012] Assays for determining proteolytic activity typically include the step of determining the extent to which the test substrate is converted into its cleavage products. After the polypeptide contacts the test substrate, the production of one or more cleavage products or an increase in the amount of cleavage products is observed, indicating that the polypeptide has proteolytic activity. The test step may involve comparison of the substrate with the cleavage products. The comparison may involve determining the amount of the substrate and / or the amount of one or more cleavage products. In addition, assays for determining proteolytic activity may include the step of comparing a test sample with a reference sample, where the reference sample typically contains (a) a polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 1 and known to have proteolytic activity, and (b) a test substrate known to be cleaved by the polypeptide of (a). In one aspect, the assay for determining proteolytic activity includes separating the substrate from the cleavage products by electrophoresis or column chromatography and (optionally) spectroscopic analysis. Conveniently, the test substrate is labeled with one or more markers to more easily detect the decrease in the test substrate and / or the increase in the product. As used herein, the term "label / marker" refers to a detectable marker and includes, for example, radiolabels, antibodies, fluorescent markers. The amount of the test substrate and / or cleavage products can be determined by, for example, autoradiographic techniques or spectroscopy, including methods based on energy resonance transfer between at least two markers. Alternatively, immunological methods such as western blotting or ELISA can be utilized for detection. A preferred method for determining the proteolytic activity of the polypeptides of the present invention is described in the examples below for illustration of the practice of the present invention. In a particularly preferred embodiment of the present invention, at 37 °C, within 120 minutes, a buffer selected from 100 mM Tris-HCl, pH 8.0 or PBS (50 mM Na 2 HPO 4 , 150 mM NaCl, pH 7.4) is used. If more than 20%, preferably more than 95%, of the test substrate is converted into cleavage products (such as light and heavy chains), the polypeptide has proteolytic activity. If the test substrate is not a full-length neurotoxin but, for example, a fragment of the full-length neurotoxin or a derivative of the neurotoxin, the same conditions still apply. Obviously, the cleavage products will be different at this time. However, the skilled person can still quantify the corresponding cleavage products. In another aspect, the assay typically employs 100 ng of the polypeptide having proteolytic activity and a molar ratio of 1:100 relative to the substrate. In another aspect, sampling can be performed at intervals to understand the catalytic activity at different times. The assay can be improved by using, for example, multiple amounts of the polypeptide having proteolytic activity.

[0013] SEQ ID NO: 2 shows the polypeptide sequence of a polypeptide derived from Clostridium botulinum strain ATCC 3502 (GenBank accession number No: "CAL82988.1") that has no proteolytic activity and has an amino acid length of 581 residues. SEQ ID NO: 1 shows a proteolytically active derivative of SEQ ID NO: 2 that lacks amino acid residues 1 to 248 of SEQ ID NO: 2.

[0014] The term "polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 1" refers to a polypeptide that has at least 50% sequence identity to the sequence of SEQ ID NO: 1. In addition, the term refers to a polypeptide that comprises a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 1. The polypeptide may have other amino acids, such as at internal positions or at the N or C terminus of the sequence shown in SEQ ID NO: 1, or at internal positions or at the N or C terminus of an amino acid sequence having at least 50% identity to the sequence of SEQ ID NO: 1, where methionine may be present at the N terminus of the polypeptide. In addition, the term refers to a polypeptide that lacks one or more amino acid residues at internal positions or at the N or C terminus of the sequence shown in SEQ ID NO: 1 or at internal positions or at the N or C terminus of a sequence having at least 50% identity to the sequence of SEQ ID NO: 1.

[0015] As used herein, the term "sequence identity" refers to the identity between a reference amino acid sequence and a sequence under study, where the sequences are aligned to obtain the highest level of match, and the identity can be calculated using publicly available techniques or methods encoded in computer programs, such as BLASTP, BLASTN, FASTA (Altschul 1990, J Mol Biol 215:403). In one aspect, the percentage value of identity is calculated for the entire amino acid sequence. In another aspect, sequence identity is calculated for sequence lengths of up to 50 aa residues, up to 100 aa residues, up to 150 aa, up to 250 aa, 300 aa, 350 aa, 400 aa, 450 aa, 500 aa or 550 aa residues. In another aspect, sequence identity is calculated for at least 50 aa residues, at least 100 aa, at least 150 aa or at least 250 aa residues. In a preferred embodiment, sequence identity is determined for the entire length of SEQ ID NO: 1 or 2, i.e., for lengths of 333 aa or 581 aa, respectively. A series of programs based on various algorithms are available to those skilled in the art for comparing different sequences. In this case, the algorithms of Needleman and Wunsch or Smith and Waterman produce particularly reliable results. To perform sequence alignment and calculate the identity values of the sequences shown herein, a commercial program DNASTAR Lasergene MegAlign version 7.1.0 based on the Clustal W algorithm is used for the entire sequence region, with the following settings: Pairwise alignment parameters: Gap penalty: 10.00, Gap length penalty: 0.10, Protein weight matrix: Gonnet250. Unless otherwise specified, the above settings are generally used as the standard settings for sequence alignment.

[0016] As used herein, the term "at least 50% sequence identity" refers to at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%.

[0017] The polypeptide with proteolytic activity of the present invention may have the same number of amino acids as the reference polypeptide sequence shown in SEQ ID NO: 1. The present invention also includes polypeptides with additional or fewer amino acid residues. In one aspect, the polypeptide with proteolytic activity of the present invention is or comprises a truncated mutant of SEQ ID NO: 1 or 2 or a truncated mutant of a polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 1 or 2. The truncated mutant of SEQ ID NO: 2 may lack one or more amino acid residues at the N-terminus, for example, at amino acid position 249. The truncated mutant may be an N-terminal or C-terminal truncated mutant and / or an internal truncated mutant having proteolytic activity. In one aspect, this truncated mutant of SEQ ID NO: 2 lacks amino acids 1 to 248 of SEQ ID NO: 2. In another aspect, the truncated mutant of SEQ ID NO: 2 is a C-terminal truncated mutant. In one aspect, the indicated truncated mutant lacks up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 150 or up to 170 consecutive amino acid residues. In another aspect, the polypeptide with proteolytic activity of the present invention has an amino acid length of at least 200 aa residues, at least 250 aa residues, at least 300 aa residues or at least 333 aa residues. In another aspect, the polypeptide with proteolytic activity of the present invention has at most 333 aa residues, at most 350 aa residues, at most 573 residues, at most 581 aa residues, at most 592 aa residues, at most 600 aa or at most 617 aa residues.

[0018] In another aspect, the polypeptide with proteolytic activity of the present invention encompasses: polypeptides containing additional amino acid residues at the N- or C-terminus and / or internal positions of the polypeptide chain of SEQ ID NO: 1 (or a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1). These additional amino acid residues may contain up to 5, up to 10 or even up to 200, 300 or up to 400 consecutive amino acid residues. In one aspect, the indicated additional amino acid residues serve as inhibitors of proteolytic activity. In another aspect, the indicated additional amino acid residues can be removed by a protease. In another aspect, additional residues that inhibit the proteolytic activity of the polypeptide of the present invention are excluded. The additional amino acid residues may flank one or more protease cleavage sites. In another aspect, the additional amino acid sequence can serve as a detectable tag and / or allow binding to a solid support.

[0019] In another aspect, the polypeptide chain of SEQ ID NO: 1 (or a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1) is modified by exchanging one or more amino acid residues. As used herein, the term "exchange" refers to replacing one amino acid with a different amino acid. For example, up to 1aa, 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 15aa, 20aa or up to 50aa within the polypeptide sequence may be replaced. The exchange may involve conservative or non-conservative amino acid changes, for the purpose of, for example, increasing or decreasing the substrate binding or proteolytic activity of the polypeptide of the present invention.

[0020] In one aspect, the proteolytically active polypeptides of the present invention encompass: polypeptides that can hydrolyze a substrate into two or more natural cleavage products. In another aspect, the polypeptides of the present invention hydrolyze the substrate into two or more cleavage products different from the natural cleavage products. As used herein, the term "natural cleavage product" or "natural product" refers to a product derived from the substrate having the same amino acid sequence when compared to the product produced from the same substrate in a wild-type cell culture. In one aspect, the cleavage product is a double-stranded neurotoxin of botulinum neurotoxin or tetanus neurotoxin. In another aspect, the double-stranded neurotoxin is a neurotoxin isolated from Clostridium botulinum serotypes A, B, C1, D, E, F or G. In other aspects, the double-stranded neurotoxin is a natural double-stranded neurotoxin.

[0021] Table 1 shows the precursors, TeNT and the natural double-stranded neurotoxins of BoNT / A-G, and identifies the exposed loops containing the amino acid sequences cleaved by the polypeptides of the present invention.

[0022]

[0023]

[0024]

[0025] It should be understood that the definitions and explanations of the above and following terms apply to all aspects described in this specification unless otherwise stated.

[0026] The polypeptide with proteolytic activity of the present invention is applicable to a variety of applications. One commercially relevant application is its use in the preparation of therapeutic neurotoxins, such as those isolated from Clostridium botulinum. Currently, cell cultures of Clostridium botulinum used to prepare commercially available botulinum neurotoxin preparations are contaminated with large proportions of untreated and / or partially treated neurotoxins, both of which negatively impact (i.e., reduce) the specific activity of these pharmaceutical compositions. By employing the proteolytically active or activated polypeptide of the present invention, for example, after lysing Clostridium botulinum, it would be possible to process compositions containing untreated and / or partially treated neurotoxins, thereby converting these contaminants into fully processed neurotoxins. Thus, commercially available products with increased specific activity of the neurotoxin can be provided, where the total amount of bacterial proteins can be reduced, further decreasing the risk of antibody formation in patients.

[0027] In another aspect, the present invention relates to a nucleic acid molecule that comprises a nucleic acid sequence encoding the polypeptide of the present invention and optionally regulatory elements. As used herein, the term "regulatory element" refers to elements that regulate gene expression (including transcription and translation), and includes, for example, tata box, promoter, enhancer, ribosome binding site, Shine - Dalgarno sequence, IRES region, polyadenylation signal, terminal capping structure, etc. The regulatory elements can comprise one or more heterologous regulatory elements or one or more homologous regulatory elements. A "homologous regulatory element" is a regulatory element of the wild - type cell from which the nucleic acid molecule of the present invention is derived, and it is involved in the regulation of gene expression of the nucleic acid molecule or the polypeptide in that wild - type cell. The present invention also encompasses nucleic acid molecules that comprise heterologous regulatory elements. The term "heterologous regulatory element" is a regulatory element that is not involved in the regulation of gene expression of the nucleic acid molecule or the polypeptide in the wild - type cell. Also included are regulatory elements for inducible expression, such as inducible promoters. The nucleic acid molecule can be, for example, hnRNA, mRNA, RNA, DNA, PNA, LNA, and / or modified nucleic acid molecules. The nucleic acid molecule can be circular, linear, integrated into the genome, or episomal nucleic acid. Also encompassed are tandem constructs encoding fusion proteins that comprise 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides of the present invention. Additionally, the nucleic acid molecule can comprise a sequence encoding a signal sequence for intracellular trafficking, such as a signal for trafficking into intracellular compartments or for trafficking across the cell membrane.

[0028] In another aspect, the present invention relates to a vector comprising a nucleic acid molecule based on the nucleic acid molecule of the present invention. The vector may be suitable for in vitro and / or in vivo expression of the polypeptide of the present invention. The vector may be a transient and / or stable gene expression vector. In one embodiment, the vector further comprises a regulatory element and / or a selection marker. In one embodiment, the vector is a virus-derived vector, in another embodiment, it is a phage-derived vector, and in another embodiment, it is a bacterium-derived vector.

[0029] In another aspect, the invention relates to a cell comprising the nucleic acid molecule or vector of the invention. As used herein, the term "cell" encompasses prokaryotic cells and / or eukaryotic cells for expressing the nucleic acid molecule or the vector and in particular the polypeptide of the invention. The cell may be a host cell that does not express the polypeptide of the invention or its homolog. As used herein, the term "homolog" refers to a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. However, the invention also encompasses cells expressing the polypeptide of the invention or its homolog, particularly wild-type cells. In certain aspects, the cells of the invention are selected from Clostridium botulinum, Clostridium butyricum, Clostridium baratii, and Clostridium tetani. In a preferred aspect, the cell is Clostridium botulinum serotype A, B, or F. In another aspect, the cell is the Hall strain of Clostridium botulinum (ATCC 3502). In another aspect, the cell is the BoNT / A-producing strain ATCC 19397 of Clostridium botulinum, which is also known as NCTC 4587 and NCTC 7272. In another aspect, the cell is the BoNT / A-producing strain NCTC 2916 of Clostridium botulinum. In another aspect, the cell is the BoNT / A2-producing strain Kyoto-F and Mauritius / NCTC 9837 of Clostridium botulinum. In another aspect, the cell is the BoNT / A3-producing strain A254 Loch Maree / NCTC 2012 of Clostridium botulinum. In another aspect, the cell is the BoNT / A4- and B-producing strain CDC657 of Clostridium botulinum. In another aspect, the cell is the BoNT / A5- and B3'-producing strain H04402 065 of Clostridium botulinum. In another aspect, the cell is the BoNT / B1-producing strain Okra / NCTC 7273 of Clostridium botulinum. In another aspect, the cell is the BoNT / B- and F-producing strain CDC4013 / NCTC 12265 of Clostridium botulinum. In another aspect, the cell is the BoNT / F1-producing strain Langeland / NCTC 10281 of Clostridium botulinum.In another aspect, the cell is Clostridium sporogenes, Clostridium perfringens, Clostridium acetobutylicum, B. cereus, B. thuringiensis, B. mycoidis, B. thermoproteolyticus, B. anthracis, B. megaterium, B. subtilis, E. coli or a yeast cell. In one aspect, the polypeptide of the present invention is modified in the cell (i.e., glycosylated, phosphorylated, protease-treated, etc.). The modification also includes the addition of non-protein cofactors, including metal ions. Cells containing the non-proteolytically active polypeptide as described above, any intermediate polypeptide products, and the ultimately proteolytically active polypeptide described herein are included in the present invention. The present invention also encompasses cells containing an expression inducer of the polypeptide of the present invention. The expression inducer can be a nucleic acid molecule, a polypeptide, or a chemical entity, including small chemical entities having the effect of increasing the amount or activity of the proteolytically active polypeptide of the present invention in a cell culture or its lysate. The expression inducer is capable of, for example, increasing the transcription or translation of a nucleic acid molecule encoding the polypeptide of the present invention. Alternatively, the expression inducer can be a compound capable of activating the non-proteolytically active polypeptide SEQ ID NO: 2 (or a polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 2). In one aspect, the cell contains an inducer, which is a proteolytically active polypeptide capable of removing the inhibitory amino acid residues at the N-terminus of the polypeptide. The inducer can be expressed, for example, by recombinant methods known to those skilled in the art. Alternatively, the inducer can be isolated from the cell, such as a Clostridium cell.

[0030] The present invention also relates to the use of the nucleic acid molecule of the present invention for the preparation of the proteolytically active polypeptide of the present invention.

[0031] In a related aspect, the present invention relates to a method for preparing a proteolytically active polypeptide, the method comprising the steps of: (a) chemically synthesizing or translating from a nucleotide sequence a polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 1; and (b) purifying the polypeptide of step (a).

[0032] The term "chemical synthesis" refers to the synthesis of polypeptides by chemical means. The methods are reviewed, for example, in Nilsson et al., Ann. Rev. Biophys. Biomol. Struct. 2005. 34:91–118. The term "purified polypeptide" refers to the removal of compounds other than the polypeptide from a mixture containing the polypeptide of the invention. This is also used to mean the removal of the polypeptide of the invention from a mixture containing compounds other than the polypeptide of the invention. In certain aspects, the term refers to the isolation of a proteolytically active polypeptide from a precursor that is not proteolytically active.

[0033] Nucleic acids can be translated in cells or cell-free systems. A person skilled in the art can employ a variety of cell-free translation systems. The present invention encompasses, for example, the translation in a cell-free protein translation system containing rabbit reticulocyte lysate, wheat germ lysate, Escherichia coli lysate, or other cell lysates, such as lysates produced from Clostridium botulinum. The present invention also encompasses the translation of the polypeptide of the invention from the nucleotide sequence of the invention or the vector of the invention. Transcription can be regulated or controlled by one or more heterologous regulatory elements or homologous regulatory elements. This aspect of the present invention also encompasses translation in wild-type cells, i.e., cells isolated from nature, such as Clostridium botulinum, Clostridium butyricum, Clostridium baratii, and Clostridium tetani. In a particular aspect, the cell is the Hall strain of Clostridium botulinum (ATCC 3502). A variety of standard means and methods can be employed by a person skilled in the art to introduce a nucleic acid molecule or vector into a cell and express the polypeptide of the invention as a recombinant protein in the cell. In addition, a person skilled in the art should be aware of many standard techniques for isolating polypeptides from cells or cell lysates or from cell-free expression systems (e.g., Recombinant DNA Principles and Methodologies, J. Green, Marcel Dekker Inc., 1998; The Condensed Protocols: From Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2006; Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2000). Any of these means and methods can be used in the methods of the present invention.

[0034] The first polypeptide of the present invention can be translated from a nucleic acid molecule encoding the polypeptide having proteolytic activity. SEQ ID NO: 26 is an example of such a nucleic acid molecule. Alternatively, the nucleic acid molecule can encode a precursor polypeptide that does not have proteolytic activity, but this precursor polypeptide can be converted into the polypeptide having proteolytic activity of the present invention. SEQ ID NO: 27 is an example of the nucleic acid molecule. The precursor polypeptide without proteolytic activity is also referred to as "inactive BoNT hydrolase", abbreviated as iBH. The polypeptide having proteolytic activity can be activated during or after translation, for example, or by contacting the polypeptide without proteolytic activity with a protease that can remove the inactivating amino acid residues at the N-terminus of the polypeptide without proteolytic activity. An example of the polypeptide without proteolytic activity is the polypeptide shown by SEQ ID NO: 2. Another example is a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 2. In one aspect, the term "inactivating amino acid residues at the N-terminus" refers to the first 248 aa residues of the polypeptide. In another aspect, the term refers to a fragment of the polypeptide shown having up to 10 aa, 50 aa, 100 aa, 150 aa, 200 aa, 250 aa residues. Any of these polypeptides can be used in the method of the present invention to prepare a polypeptide having proteolytic activity. In one aspect, the protease capable of removing the inactivating amino acid residues from the N-terminus of this polypeptide is isolated from, for example, Clostridium botulinum, Clostridium butyricum, Clostridium baratii, and Clostridium tetani. In another aspect, the protease capable of removing the inactivating amino acids is provided by providing a fractionated lysate or an unfractionated lysate of the cell. The inactivating amino acid residues can be removed by contacting the polypeptide without proteolytic activity with the lysate and culturing until the polypeptide without proteolytic activity is converted into a polypeptide having proteolytic activity.

[0035] In another aspect of the method of the present invention, the polypeptide is translated in a cell. The cell can be a prokaryotic or eukaryotic cell. In one aspect, the cell is selected from Escherichia coli, Bacillus subtilis, or yeast. The present invention also encompasses translating the polypeptide of the present invention in a wild-type cell (i.e., a cell isolated from nature), such as any known isolate of Clostridium botulinum, Clostridium butyricum, Clostridium baratii, and Clostridium tetani. In a specific aspect, the cell is the Hall strain of Clostridium botulinum (ATCC 3502). In another specific aspect, the cell is the cell of the present invention as described above.

[0036] The translation products obtained by the method of the present invention can be purified by a variety of means, which are all known to those skilled in the art (for example, Recombinant DNA Principles and Methodologies, J. Green, Marcel Dekker Inc., 1998; The Condensed Protocols: From Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2006; Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2000). Typical methods for purifying the polypeptides of the present invention may include centrifuging cell lysates, ammonium sulfate precipitation of proteins, resuspending the proteins, centrifuging the resuspended proteins, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, etc. Combinations of several different sequences of the said steps can be used to purify the polypeptides of the present invention. The preferred methods for purifying the polypeptides of the present invention are described in the examples of the present invention.

[0037] In one aspect, the purification step comprises binding the polypeptides of the present invention to a solid support. The term "solid support" refers to a matrix that comprises, for example, silica, cross-linked dextran, cross-linked polyacrylamide or cross-linked agarose, etc. It also specifically includes polypeptides, glass, polystyrene, polypropylene, polyethylene, polyethylene glycol (PEG), dextran, nylon, amylase, natural or modified cellulose, polyacrylamide, gabbro and magnetite. In one aspect of the present invention, the solid support is a polysaccharide matrix selected from the group consisting of agarose gel (sepharose), dextran gel, agarose, dextran cellulose (sephacell), microcrystalline cellulose and alginate beads. In another aspect, the solid support may consist of glass beads and / or a polypeptide matrix.

[0038] In one aspect, the solid support is linked to an antibody of the present invention. In one aspect, the term "linked" refers to a stable connection or stable coupling. In another aspect, the linkage includes, for example, indirect or direct, irreversible or reversible, physicochemical, electrostatic, and / or covalent bond interactions. In one aspect, the antibody is covalently linked to the solid support either directly or via a linker molecule. The antibody can be linked to the solid support via a linker, which includes small molecule compounds and peptide (or polypeptide) linker molecules. The solid support can have almost any possible structural configuration or arrangement as long as the coupled antibody can bind to its antigen. Thus, the matrix or solid support can be spherical (such as a bead) or cylindrical (such as the inner surface of a test tube or the outer surface of a rod). Alternatively, the surface can be irregular or planar, such as a thin plate or a test strip.

[0039] The antibody linked to the solid support can be used, for example, in the preparation method or diagnostic method of the present invention. In one aspect, the preparation method can include a step of affinity chromatography, wherein the affinity chromatography is based on the antibody linked to the solid support. In one embodiment, the antibody is an antibody that specifically binds to a polypeptide with proteolytic activity of the present invention. In other embodiments, the possible solids are shown for antibodies that specifically bind to polypeptides without proteolytic activity of the present invention.

[0040] In another aspect, a method for preparing a polypeptide with proteolytic activity of the present invention includes: purifying the polypeptide of the present invention from a mixture containing additional components. The purification can be based on, for example, polarity, charge, and size. Thus, the method can include, in one aspect, one or more separation steps selected from the group consisting of: normal-phase HPLC, reverse-phase HPLC, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC) (including anion exchange chromatography and cation exchange chromatography), size exclusion chromatography (SEC), and gel permeation chromatography (GPC).

[0041] In another aspect, the purification includes the following steps: (a) separation by ion exchange chromatography; (b) separation by size exclusion chromatography; (c) separation by hydrophobic interaction chromatography; and (d) separation by size exclusion chromatography.

[0042] One or more components collected from the chromatographic column can be concentrated by, for example, precipitation or ultrafiltration.

[0043] In one aspect, the present invention relates to a composition comprising a polypeptide with proteolytic activity of the present invention. By the method described herein, a polypeptide with proteolytic activity of the present invention can be prepared, which is substantially free of polypeptides without proteolytic activity. In other words, the method of the present invention provides a composition of a polypeptide with proteolytic activity and substantially free of contamination by an inactive precursor protein of the polypeptide of the present invention. It is considered that the composition is substantially free of contamination or substantially free of precursor polypeptides without proteolytic activity. If a Western blot-based detection method is used, less than 5% of the precursor without proteolytic activity can be detected, where the 5% refers to the amount of the precursor without proteolytic activity relative to the total amount of the polypeptide with proteolytic activity and the inactive polypeptide. In another aspect, the composition is substantially pure and comprises at least 50% of the polypeptide with proteolytic activity of the present invention, where the 50% refers to the amount of the precursor with proteolytic activity relative to the total amount of proteins contained in the composition. In another aspect, the substantially pure composition comprises at least 75%, 80%, 90% or at least 98% of the polypeptide with proteolytic activity.

[0044] In another aspect, the present invention also relates to a polypeptide obtainable from the method for preparing a polypeptide with proteolytic activity described above and in the examples. In one aspect, the polypeptide with proteolytic activity is a polypeptide with proteolytic activity containing the polypeptide sequence of SEQ ID NO: 1. In another aspect, the polypeptide with proteolytic activity is a polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 1. In another aspect, the polypeptide with proteolytic activity is a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. As used herein, the term "obtainable polypeptide" in one aspect refers to a polypeptide translated from the nucleic acid of the present invention. The polypeptide can then be post-translationally modified, such as acylation, alkylation, amidation, amino acid addition, amino acid deletion, glycosylation, oxidation, S-glutathionylation, phosphorylation, sulfation, proteolytic processing, etc. In addition, the polypeptide can bind to metal ions, such as Li + 、Na + 、K + 、Ag + 、Cs + 、Mg 2+ 、Ca 2+ 、Co 2+ 、Ni 2+ 、Mn 2+ 、Cu 2+ or Zn 2+ . Preferably, the metal ion is Zn 2+ 、Mn 2+ or Co 2+ .

[0045] In one aspect, the invention also relates to an antibody that specifically binds to the polypeptide of the invention. As used herein, the term "antibody" encompasses monoclonal antibodies, polyclonal antibodies, single-chain antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, bispecific antibodies, synthetic antibodies, chemically or enzymatically modified derivatives, fragments of any of the foregoing antibodies or aptamers composed of naturally occurring and / or chemically modified nucleic acids. Fragments of the antibody include F(ab')2, F(ab), Fv or scFv fragments or chemically or enzymatically modified derivatives of any of the foregoing fragments.

[0046] In one aspect, the antibody of the invention will specifically bind to the proteolytically active polypeptide of the invention or its non-proteolytically active precursor. In one aspect, an antibody specific for the non-proteolytically active polypeptide of the invention cross-reacts with the non-proteolytically active polypeptide described herein. In another aspect, the antibody can distinguish the proteolytically active polypeptide of the invention from its inactive precursor. In another aspect, the epitope specific for the antibody is located in the amino acid region of the non-proteolytically active polypeptide but is not present in the proteolytically active polypeptide. For example, the epitope may be an epitope of the polypeptide region composed of amino acid residues 1 to 248 of the polypeptide, and the polypeptide comprises a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 2.

[0047] In another aspect, the epitope is formed by amino acid residues located at the N-terminus of amino acid 248 of the polypeptide, and the polypeptide comprises a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 2. In another aspect, the epitope is removed by proteolytic treatment from the non-proteolytically active polypeptide described herein.

[0048] In another aspect, the epitope specific for the antibody of the invention is an epitope located at the N-terminus of the polypeptide, and the polypeptide comprises a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. As used in one aspect of the invention, the term "N-terminus" refers to a polypeptide region comprising the N-terminal 50 amino acid residues of the polypeptide sequence, preferably the N-terminal 25 amino acid residues of the polypeptide sequence. In a specific aspect, the term refers to the N-terminal 14 amino acid residues. As used herein, the term "epitope" refers to the antigenic determinant recognized by the antibody of the invention. In one aspect, the epitope is a linear epitope, and in another aspect, the epitope is a conformational epitope. In a specific aspect, the antigenic determinant consists of a peptide of the amino acid sequence at the N-terminus of the proteolytically active polypeptide of the invention, and the peptide may have a length of 7 to 14 amino acids, preferably 8, 9, 10, 11, 12, 13 or 14 amino acid residues.

[0049] In one aspect, the term "specifically binds" or "specifically binds to" means that the antibodies of the present invention do not cross-react to a significant extent with other epitopes on the polypeptides of the present invention or other general polypeptides. Epitope specificity is an important feature of the antibodies of the present invention. The specificity of the antibodies for polypeptides with and without proteolytic activity should be at least 95%, at least 96%, at least 97%, at least 98%, at least 99% in one aspect. Specific binding can be determined by a variety of well-known techniques, including, for example, competition assays. Another important feature is the sensitivity of the antibody. In one aspect of the present invention, the sensitivity is such that at least 70%, at least 80%, at least 90%, at least 95% of the epitopes contained in the sample are bound. Sensitivity can be determined by well-known techniques. Those skilled in the art will be able to determine the operation and ideal test conditions for each assay by routine experimentation. Traditional techniques for binding studies include radioimmunoassay, ELISA, equilibrium dialysis, isothermal microcalorimetry, assays (surface plasmon resonance, SPR) or other surface adsorption methods. The SPR system detects antibody-antigen interactions. The SPR response reflects the change in mass concentration at the detector surface when the analyte binds or dissociates. Based on SPR, real-time measurement results directly monitor the interaction occurring immediately, see BIAapplications Handbook, AB Edition (reprinted 1998), No.: BR-1001-86; BIAtechnology Handbook, AB Edition (reprinted 1998), No.: BR-1001-84. Binding characteristics (such as the sensitivity of the antibodies of the present invention) can in principle be determined by, for example, binding assays using a solid antigen (ligand) present on the sensor surface. The antibody to be tested (analyte) will be provided in a mobile phase (i.e., a solution). In some cases, the antigen is indirectly linked to the surface by binding to another immobilized molecule called a capture molecule. When the antibody is injected in discrete pulses over a surface with immobilized antigen, three phases can be essentially distinguished: (i) binding of the antibody to the antigen during sample injection; (ii) an equilibrium or steady state during sample injection, where the rate of antibody binding is balanced with the rate of dissociation from the antibody-antigen complex; (iii) dissociation of the antibody from the surface during buffer flow. It should be understood that the analysis can alternatively be carried out by immobilizing the antibody to be tested and using a solution containing the antigen as the mobile phase. The binding and dissociation phases provide kinetic information (k a and k d , the rates of complex formation and dissociation, kd / k a = K D )。The equilibrium phase provides information on the affinity of the analyte-ligand interaction (K D ). In one aspect of the invention, the antibodies of the invention have a K of less than 0.5 μM D , and in another aspect, a K of less than 0.05 μM D , and in yet another aspect, a K of less than 0.02 μM D .

[0050] The antibodies of the invention can be made by using methods such as those described in Harlow and Lane, 1988 (Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988). Monoclonal antibodies can be prepared by the techniques initially described in Kohler and Milstein, 1975 (Kohler and Milstein 1975, Nature 256:495) and Galfre and Milstein, 1981 (Galfre and Milstein 1981, Meth Enzymol 73:3). The techniques include fusing murine myeloma cells and spleen cells derived from immunized mammals. The antibodies can be further improved by techniques well known in the art. For example, surface plasmon resonance used in the system can be used to increase the efficiency of phage antibodies that bind to the foregoing epitopes within the polypeptides of the invention (see Schier et al., 1996, Human Antibodies Hybridomas 7:97; Malmborg et al., 1995, J. Immunol Methods 183:7).

[0051] In one aspect of the present invention, the antibody is generated by using a peptide comprising or consisting of the aforementioned epitope. The peptide can be prepared, for example, by synthesis or by recombinant expression. Alternatively, the antibody of the present invention can be prepared by using a naturally occurring polypeptide of the present invention with or without proteolytic activity. In the latter case, it should be understood that the formed antibody will be further tested for its specificity to the polypeptide of the present invention. In other aspects of the present invention, the monoclonal antibody of the present invention is prepared by using the polypeptide of the present invention, wherein the polypeptide of the present invention can be treated with a detergent to make the epitope immunogenic available. However, it should be understood that if the antibody is to target a conformational epitope, the detergent treatment should not be employed. In another aspect, an immunostimulant such as keyhole limpet hemocyanin (KLH) can also be applied to the method, especially when using a synthetic peptide.

[0052] The antibody of the present invention can be used, for example, in affinity chromatography, immunoprecipitation and immunolocalization of the polypeptide of the present invention, and for monitoring the presence of the polypeptide in a sample or a recombinant organism. In addition, the antibody of the present invention can be used in a detection method or a diagnostic method. In a specific aspect, the antibody of the present invention is used in western blotting or ELISA. In addition, the antibody of the present invention can be used in therapeutic applications. Specifically, the antibody can be used to inhibit the activity of the polypeptide with proteolytic activity of the present invention. Therefore, the antibody of the present invention also has many therapeutic applications as described below.

[0053] The present invention also relates to the application of the polypeptide with proteolytic activity of the present invention in a method for proteolytically treating a polypeptide. In one aspect, the present invention relates to a method for preparing a proteolytically treated polypeptide, the method comprising the step of contacting (a) with (b): (a) a first polypeptide, which is the polypeptide of the present invention, and (b) a second polypeptide, which is susceptible to proteolysis by the first polypeptide, wherein the contacting results in the proteolytic treatment of the second polypeptide into at least two cleavage products.

[0054] The present invention also relates to the use of dLys-N and / or Lys-C and / or spermidyl endopeptidase (endoproteinase Arg-C, LeR) from Lysobacter enzymogenes (ATCC 29487) (Wright DS, Graham LD, Jennings PA. Biochim Biophys Acta. Dec 22, 1998;1443(3):369-74). In addition, the present invention also encompasses the use of plasmin and / or Omptin protein (OmpT), a membrane-bound serine protease that cleaves at the (Arg / Lys)-(Arg / Lys) motif (K. Sugimura and T. Nishihara. J. Bacteriol. 170 (1988), pp. 5625–5632), in a method for proteolytically treating CNTs (such as BoNT / A). In one aspect, the present invention relates to a method for preparing a proteolytically treated polypeptide, the method comprising the step of contacting (a) with (b): (a) a first polypeptide, which is Lys-C or Lys-N, and (b) a second polypeptide, which is susceptible to proteolysis by the first polypeptide, wherein the contacting results in the proteolytic treatment of the second polypeptide into at least two cleavage products, and wherein the second polypeptide is the single chain of BoNT / A. The term "Lys-C" refers to the 33 kDa serine endopeptidase Lys-C (lysyl endopeptidase, LeK, Genbank accession number Q7M135) from Lysobacter enzymogenes that specifically cleaves the peptide bond at the C-terminus of lysine or a homolog thereof having at least 60% sequence identity. The term "Lys-N" refers to the metalloendopeptidase Lys-N isolated from Grifola frondosa and Pleurotus ostreatus (Nonaka T et al., 1997, J Biol Chem. 272:30032-30039; Nonaka T et al., 1998, J Biochem. 1998 124:157-162; Hori T et al., 2001, Acta Crystallogr D Biol Crystallogr. 57:361-368). The term also encompasses homologs of the protease having at least 60% sequence identity.

[0055] The method can also be used, for example, to prepare proteolytically processed neurotoxins (CNT) or botulinum neurotoxins (BoNT). The term "BoNT" as used throughout this invention refers to botulinum neurotoxin and refers to the neurotoxin obtainable from Clostridium botulinum, such as BoNT of serotypes A, B, C1, D, E, F, or G. The terms "CNT" and "BoNT" also encompass recombinant neurotoxins and encompass one or more modifications (including chemical modifications or genetic modifications). The term "genetic modification" refers to the deletion, substitution, or addition of one or more amino acid residues. Using the method of the present invention, a neurotoxin composition contaminated with significantly less unprocessed or partially processed neurotoxin can be obtained because these contaminants are effectively processed into double-stranded neurotoxins. In one aspect, the double-stranded neurotoxin is a natural double-stranded neurotoxin, wherein the C-terminus of the light chain and the N-terminus of the heavy chain are identical to the corresponding fully processed double-stranded neurotoxin isolated from wild-type Clostridium botulinum.

[0056] The term "contact" as used herein means bringing at least two different compounds into physical proximity to allow physical and / or chemical interactions between these compounds. According to the method of the present invention, in one aspect, the two different compounds are a first polypeptide and a second polypeptide contained in a solution. The contact is carried out under conditions and for a time sufficient to allow the first polypeptide and the second polypeptide to interact. The term "proteolytically processed polypeptide" as used herein refers, in one aspect, to a polypeptide in which one or more peptide bonds of the polypeptide chain have been hydrolyzed or cleaved. In another aspect, the term refers to a polypeptide that has been proteolytically cleaved by an endoprotease or endopeptidase. In another aspect, the term refers to a polypeptide that has been cleaved to an extent of at least 50%. In another aspect, the proteolytically processed polypeptide is the second polypeptide. In another aspect, at least 60%, 70%, 80%, 90%, or 95% is proteolytically processed.

[0057] As used herein, the term "first polypeptide" refers to the polypeptide of the present invention, i.e., a polypeptide having proteolytic activity or activation, also known as "active BoNT hydrolase". Since this active BoNT hydrolase can be obtained from the supernatant of Clostridium botulinum, it was initially named natural BoNT hydrolase, abbreviated as "nBH". However, the terms "first polypeptide" and "nBH" also refer to BoNT hydrolases that can be obtained from other sources. As used herein, the term "second polypeptide" refers to the substrate of the first polypeptide. The term "proteolytically susceptible" refers to the characteristic or condition of the second polypeptide, and is used herein to mean that the second polypeptide can be proteolytically cleaved by the first polypeptide. In other words, the term "proteolytically susceptible" means that the second polypeptide contains protease recognition and cleavage sites that allow it to serve as a substrate for the first polypeptide. The "second polypeptide" is the substrate of the first polypeptide and is proteolytically processed into two or more cleavage products. Using the above assays, one skilled in the art can determine whether a given polypeptide is a substrate of the first polypeptide, i.e., a "second polypeptide", according to the definition of the present invention. The term "at least two cleavage products" includes, for example, up to 2, 3, 4, 5, and up to 6 cleavage products.

[0058] The method can be used, for example, to prepare a pharmaceutical composition containing a Clostridium botulinum neurotoxin or to generate polypeptide fragments used in mass spectrometry. The first polypeptide and the second polypeptide can be contacted at different steps in a method for preparing a proteolytically processed polypeptide. In one aspect, the step of contacting the first polypeptide and the second polypeptide is carried out intracellularly. In a specific aspect of this embodiment, the first polypeptide and the second polypeptide are expressed intracellularly.

[0059] In another aspect, the contacting step is carried out in a cell lysate or a purified cell lysate. This aspect encompasses adding the first polypeptide to the lysate or the purified lysate. The first polypeptide can be added at different steps in the process of purifying the second polypeptide from the cell lysate. For example, the first polypeptide can be added before or after the following steps: protein precipitation, ion exchange chromatography, hydrophobic interaction chromatography, and / or size exclusion chromatography. In addition, adding the first polypeptide to a pharmaceutical composition is also encompassed. In this aspect, the polypeptide of the present invention is used, for example, to proteolytically cleave the second polypeptide to, for example, activate the second polypeptide as a therapeutic agent contained in a pharmaceutical composition. It is also contemplated to administer the first polypeptide to a subject to proteolytically process the second polypeptide within the subject. Administration also includes co-administering the first polypeptide and the second polypeptide. The method also includes a step of incubating under conditions and for a time sufficient to cleave the second polypeptide. In one aspect, the conditions can include adding a buffer selected from the group consisting of: 100 mM Tris-HCl, pH 8.0 or PBS (50 mM Na 2 HPO 4, 150 mM NaCl, pH 7.4). Preferred buffer conditions include 100 mM Tris-HCl, pH 8.0. The "time sufficient for cleavage" can be determined using the above assays. In one aspect, the "time sufficient for cleavage" depends on the degree of cleavage that the proteolytically treated polypeptide or composition containing it should have. In one aspect, the method includes the step of incubating the first polypeptide and the second polypeptide for at least 30 minutes, 60 minutes, 120 minutes, or at least 240 minutes. In another aspect, the first polypeptide and the second polypeptide are incubated for up to 30 minutes, 60 minutes, 120 minutes, 240 minutes, 480 minutes, or up to 600 minutes. In another aspect, the method includes the step of incubating the first and second polypeptides at 4 °C or 37 °C. In another aspect, the method includes the step of incubating for up to 1 hour, up to 2 hours, 4 hours, 6 hours, 10 hours, or up to 16 hours.

[0060] In one aspect, the polypeptide chain of the second polypeptide comprises a sequence selected from any one of SEQ ID NOs: 4 to 25. In a more specific aspect, the polypeptide chain of the second polypeptide comprises a sequence selected from any one of SEQ ID NOs: 4 to 25, and wherein the second polypeptide is cleaved at the C-terminus of the basic amino acid residue within any one of the sequences of SEQ ID NOs: 4 to 25. The sequences represent the amino acid sequences of known substrates of the proteolytically active polypeptides of the present invention. As shown herein, the substrate is cleaved at the C-terminus of the basic amino acid residue contained in the sequence. Compare the LC column and the H N column in Table 1. In a preferred aspect, the second polypeptide comprises a sequence selected from SEQ ID NOs: 4 to 10. In another preferred aspect, the second polypeptide is BoNT / A or a derivative thereof, including, for example, the polypeptide of SEQ ID NO: 3 or a derivative thereof. The term "derivative" as used in this aspect and other aspects of the present invention encompasses amino acid mutations such as addition, substitution, deletion, or truncation of one or more amino acid residues.

[0061] In one aspect, the second polypeptide comprises any one of SEQ ID NOs: 4 to 25 or a derivative of SEQ ID NO: 3, wherein the derivative has one or more point mutations and / or one or more additional amino acid residues. In another aspect, the derivative has up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15 point mutations. By using the activity assay for determining protease activity as described herein, one skilled in the art can determine whether a given derivative is processed by the proteolytically active polypeptide of the present invention. In another aspect, the derivative comprises a point mutation that changes a basic amino acid to a non-basic amino acid residue. In another aspect, the derivative has at least 50% sequence identity with any one of SEQ ID NOs: 4 to 25. In another aspect, the derivative or the polypeptide comprising the derivative is a substrate of the first polypeptide and can be proteolytically cleaved by the first polypeptide. A typical example is a derivative of SEQ ID NO: 3, which comprises one or more point mutations in, for example, the light chain or the heavy chain.

[0062] In another aspect, the second polypeptide comprises (a) a polypeptide sequence having at least 30% sequence identity with the sequence of SEQ ID NO: 3 [(BoNT / A of ATCC 3502, Genbank accession number.AAA23262)]; or (b) a polypeptide sequence selected from the group consisting of: tetanus neurotoxin, a protein of blood coagulation cascade factor X or prothrombin (factor II), pancreatic digestive enzymes (such as trypsin, chymotrypsin), pepsin, papain. At least 30% means at least 30%, at least 40%, at least 50%, at least 85%. In a specific aspect, the sequence identity of the second polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 3 is determined based on amino acids 420 to 466 of SEQ ID NO: 3. In another aspect, the sequence identity is determined based on any one of SEQ ID NOs: 4 to 25. In other words, this aspect refers to a second polypeptide comprising a polypeptide sequence that has, for example, at least 30% sequence identity with the polypeptide sequence of amino acids 420 to 466 of SEQ ID NO: 3. The polypeptide based on this definition can be obtained, for example, from Clostridium botulinum, Clostridium tetani or Clostridium sporogenes. The second polypeptide can be, for example, a naturally occurring neurotoxin (such as BoNT / A, B, C1, D, E, F or G) or a derivative thereof comprising one or more amino acid mutations (such as addition, substitution, deletion or truncation of one or more amino acid residues). Derivatives that cover, for example, the deletion of, for example, the native neurotoxin H C domain or a part thereof, or having other substitutions for the native neurotoxin H CDerivatives of amino acid residues of a domain and derivatives of an additional light chain of BoNT or another protein cargo molecule fused to the N-terminus of the light chain.

[0063] In another aspect, the second polypeptide may comprise additional amino acid residues at the N-terminus or C-terminus or an internal position. The additional amino acid residues may flank one or more protease cleavage sites. In another aspect, the additional amino acid sequence serves as a detectable tag and / or allows binding to an individual support. One example is a his tag or a GST tag. Another example is the amino acid sequence VPPTPGSAWSHPQFEK containing a Strep tag, preferably added to the C-terminus.

[0064] In a specific aspect, the second polypeptide is a polypeptide comprising a polypeptide sequence as shown in GenBank accession numbers CBZ04958.1, YP_002805603.1, ZP_02994746.1, YP_001788403.1, YP_001782718.1, ZP_02616437.1, ZP_02614241.1, YP_001392361.1, YP_001255575.1 or a homolog thereof having at least 50% sequence identity.

[0065] In another aspect, the biological activity of the second polypeptide is regulated by proteolytic cleavage. It is well known to those skilled in the art that the functions of many polypeptides can be regulated by proteolytic processing. As used herein, "regulation" means increasing or decreasing, activating or inactivating. For example, the biological activity of various Clostridium botulinum neurotoxins is increased or initiated by proteolytically processing the single-chain neurotoxin into a double-chain neurotoxin, wherein the double-chain neurotoxin consists of a polypeptide light chain and a heavy chain, which are covalently linked by a disulfide bond. The biological activity of the neurotoxin encompasses at least three different activities: The first activity is the "proteolytic activity" located in the light chain of the neurotoxin and is responsible for hydrolyzing the peptide bonds of one or more polypeptides involved in regulating cell membrane fusion. The second activity is the "translocation activity" located at the N-terminus of the heavy chain of the processed neurotoxin, which is involved in transporting the light chain through the lysosomal membrane into the cytoplasm. The third activity is the "receptor binding activity" located at the C-terminus of the heavy chain of the processed neurotoxin, which is involved in the binding of the neurotoxin to the target cell and the uptake of the neurotoxin by the target cell. In a preferred aspect, the term biological activity as used herein refers to proteolytic activity. In a more preferred aspect, the term refers to increasing proteolytic activity.

[0066] The biological activity of Clostridium botulinum neurotoxins can be detected by a variety of tests, all of which are known to those skilled in the art. These tests allow the determination of one or more of the above activities. For example, mouse LD 50The test or the mouse phrenic nerve - hemidiaphragm (MPN) assay (as described by Pearce et al. in 1994 (Pearce LB, Borodic GE, First ER, MacCallum RD (1994), Toxicol Appl Pharmacol 128:69 - 77) and by Habermann et al. in 1980 (Habermann E, Dreyer F, Bigalke H. (1980), Naunyn Schmiedebergs Arch Pharmacol. 311:33 - 40)) allows the determination of the toxic effects of a given neurotoxin preparation on a living organism or isolated neuromuscular tissue. To establish the toxic effects in the LD 50 assay, the neurotoxin must possess each of the three activities described above. In addition, there are many other tests available to determine, for example, whether a neurotoxin or the light chain of the neurotoxin has proteolytic activity. Such tests are based on, for example, exposing BoNT / A to SNAP - 25. Alternatively, a peptide representing the cleavage site of SNAP - 25 can be used, where the peptide can be labeled for easy detection. In a preferred aspect, the biological activity is determined using the MPN assay as described above.

[0067] In another aspect, the first polypeptide is activated by proteolytic treatment of an inactive precursor polypeptide, the inactive precursor polypeptide comprising a polypeptide sequence having at least 60% sequence identity with the sequence of SEQ ID NO:2. This aspect is based on the observation that a polypeptide having the polypeptide sequence of SEQ ID NO:2 is not proteolytically active, while its N - terminal truncation is proteolytically active. The present invention also contemplates the use of non - proteolytically active polypeptides in the methods described herein. The non - proteolytically active polypeptides described herein can be activated, for example, by removing the N - terminal fragment or removing the entire N - terminal comprising amino acid residues 1 - 248 of SEQ ID NO:2. In one aspect, the N - terminal is removed by a protease, and in another aspect, the N - terminal is removed by autoproteolysis of SEQ ID NO:2. 60% sequence identity refers to sequence alignment with the full - length NT02CB1447.

[0068] In another aspect, the method for preparing a proteolytically processed polypeptide of the present invention includes the step of purifying the proteolytically processed second polypeptide or at least one or two or more cleavage products thereof. The purification of BoNT / A expressed by Clostridium botulinum can be carried out substantially by the methods described in the prior art (DasGupta 1984, Toxicon 22, 415; Sathyamoorthy 1985, J Biol Chemistry 260, 10461). Specifically, the purification of the neurotoxin can include one or more precipitation and extraction steps, one or more concentration steps, and other different chromatographic steps. The recombinant single-chain BoNT / A and its purification are described in the prior art (Rummel et al., 2004, Mol Microbiol. 51:631-43).

[0069] In a preferred embodiment, the Clostridium strain is, for example, Clostridium botulinum that produces BoNT / A or its derivatives. For fermentation, the method described by DasGupta B.R. et al. in Toxicon, Volume 22, Issue 3, pages 414 - 424 (1984) can be used. Thus, a medium is prepared by adding 0.5% yeast extract and 0.6% autoclaved yeast paste to 2% N-Z-amine A medium, adjusting the pH to 7.2 with 4N NaOH, and then autoclaving. Another autoclaved glucose (20% weight per volume) can be added to the medium to bring the final concentration of glucose in the medium to 0.5%. Incubation can be carried out, for example, at 37°C (without stirring), where the fermentation is terminated, for example, after 96 hours. Within the scope of the present invention, in addition to the aforementioned batch fermentation, semi-batch fermentation, repeated batch fermentation, or continuous fermentation can also be carried out.

[0070] After actual fermentation and separation of the fermentation medium and cells, the fermentation medium can be subjected to one precipitation to remove large proteins. The precipitation is preferably acid precipitation. The reaction conditions for the acid precipitation are known to those skilled in the art. Usually, 1.5M H 2 SO 4, so that the supernatant is acidified to pH 3.5. Centrifugation is usually carried out at 2400x g at 4°C for 20 minutes. The precipitate obtained by centrifugation can be washed with water, preferably repeatedly. Subsequently, the precipitate can be extracted with 0.1M citric acid-trisodium citrate buffer (pH5.5), for example, for 1 hour. Subsequently, other centrifugation steps can be performed, for example, centrifugation at 9800x g at 4°C for 20 minutes. The precipitate obtained in this way can then be optionally extracted again as described above. The extracted supernatant and the secondary supernatant of the repeated extraction can then be precipitated with protamine sulfate. The precipitation can be continued overnight at 8°C, for example. Subsequently, the precipitate can be centrifuged at 12,000xg at 4°C for 20 minutes. The supernatant of the centrifugation can be treated with precipitation (for example, ammonium sulfate precipitation) to remove other larger proteins. Another centrifugation step can be added after the ammonium sulfate precipitation step, and then the precipitate obtained can be redissolved and optionally treated by dialysis. Preferably, the extract that has been dialyzed and centrifuged again can be treated with continuous chromatography steps to purify the neurotoxin. Each chromatography step is used to remove contaminants, such as protamine sulfate, remaining DNA, smaller proteins and portions of medium-sized proteins, and hemagglutinins of the botulinum neurotoxin protein complex. For this purpose, one or more chromatography steps may be used in a preferred embodiment. Optionally, the eluate of, for example, the last chromatography step may be filtered to reduce bacteria. Optionally, the eluate may be diluted and a suitable adjuvant may be added before filtration. During other steps, another sterile filtration step may be performed after the addition of the adjuvant. In one aspect, the filtration is performed in a reaction vessel, which may then be subjected to a freeze-drying step.

[0071] The present invention also relates to a composition obtainable by the method of preparing a proteolytically treated polypeptide of the present invention. In one aspect, the composition comprises a mixture of treated and untreated second polypeptides, wherein the mixture may comprise less than 5%, 4%, 3%, 2% or less than 1% of the untreated second polypeptide. In one aspect of the composition, the second polypeptide is a BoNT or a derivative thereof. The BoNT may, for example, be selected from the group consisting of serotypes A, B, C, D, E, F and G of BoNT, including derivatives thereof. The composition may be, for example, a liquid or solid composition and may comprise one or more carriers, adjuvants and / or excipients.

[0072] In another aspect, the present invention also relates to a method for preparing a drug (pharmaceutical composition), the method comprising the steps of the aforementioned method and the further step of formulating the purified double-chain neurotoxin as a drug. In one aspect, the drug comprises a mixture of treated and untreated second polypeptides, wherein the mixture may contain less than 5% of the untreated second polypeptide. In a preferred embodiment, the mixture contains less than 4%, 3%, 2% or less than 1% of the untreated second polypeptide.

[0073] The present invention also relates to various medical uses of the compounds described herein:

[0074] In one aspect, the present invention relates to the proteolytically active polypeptides of the present invention for use as a medicament or in a pharmaceutical composition.

[0075] In another aspect, the present invention relates to the compositions of the present invention for use as a medicament or in a pharmaceutical composition.

[0076] In yet another aspect, the present invention relates to the antibodies of the present invention for use as a medicament or in a pharmaceutical composition.

[0077] In yet another aspect, the present invention relates to the inhibitors of the present invention for use as a medicament or in a pharmaceutical composition.

[0078] Specifically, the present invention relates to pharmaceutical compositions comprising the polypeptides, antibodies, compositions or inhibitors of the present invention.

[0079] As used herein, the term "composition" refers to any composition formulated in solid, liquid, aerosol (or gas) form, etc. The composition comprises, for example, a therapeutically active compound of the present invention and optionally suitable auxiliary compounds, such as diluents or carriers or other ingredients. In one aspect, the therapeutically active compound is the proteolytically active polypeptide of the present invention. In another aspect, the therapeutic compound is the second polypeptide subjected to proteolytic treatment as described above, such as a double-stranded neurotoxin. In another aspect, the therapeutically active compound is the antibody of the present invention. In another aspect, the therapeutically active compound is an inhibitor of the proteolytically active polypeptide of the present invention.

[0080] In the present invention, auxiliary compounds and other ingredients are distinguished. Auxiliary compounds are those that do not cause the effects induced by the compounds of the present invention when the composition is administered for the desired purpose, while other ingredients are those that cause other effects or modulate the effects of the compounds of the present invention. Suitable diluents and / or carriers depend on the purpose for which the composition and other ingredients are to be used. Those skilled in the art can determine the suitable diluents and / or carriers without further elaboration.

[0081] The carrier must be compatible with the other components of the preparation and harmless to its recipient. The pharmaceutical carriers used may include solids, gels or liquids. Exemplary solid carriers are lactose, clay, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, etc. Examples of liquid carriers are phosphate buffered saline solutions, syrups, oils, water, emulsions, various types of wetting agents, etc. Similarly, the carrier or diluent may include time-delayed materials well known in the art, such as glyceryl monostearate or glyceryl distearate alone or in combination with waxes. The suitable carriers include those described above and other carriers well known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0082] The diluent is selected so as not to affect the biological activity of the combination. Examples of the diluent are distilled water, physiological saline, Ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or preparation may further contain other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0083] In one aspect, the pharmaceutical composition used herein comprises a bioactive neurotoxin obtained by the method of the present invention, optionally comprising one or more pharmaceutically acceptable carriers. The active neurotoxin may exist in liquid or lyophilized form. In one aspect, the compound may be present with glycerol, a protein stabilizer (e.g., human serum albumin (HSA)) or a non-protein stabilizer (e.g., polyvinylpyrrolidone or hyaluronic acid). In one aspect, the pharmaceutical composition is administered locally. The traditional routes of drug administration are intramuscular and subcutaneous (near the gland). However, depending on the nature and mode of action of the compound, the pharmaceutical composition may also be administered by other routes. The double-stranded neurotoxin polypeptide is the active ingredient of the composition and is administered in a traditional dosage form prepared by combining the drug and a standard pharmaceutical carrier according to traditional methods in one aspect. These methods may involve mixing, granulating and compressing or dissolving the appropriate ingredients into the desired preparation. It should be understood that the form and characteristics of the pharmaceutically acceptable carrier or diluent are affected by the amount of the active ingredient to be combined therewith, the route of administration and other well-known variable factors.

[0084] A therapeutically effective dose refers to the amount of the neurotoxin compound to be used in the pharmaceutical composition of the present invention for preventing, ameliorating or treating the symptoms associated with the diseases or disorders described in this specification. The therapeutic efficacy and toxicity of the compound can be determined by standard pharmaceutical methods in cell cultures or experimental animals, such as ED50 (the dose that has a therapeutic effect on 50% of the population) and LD50 (The dose that kills 50% of a population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, which can be represented by the ratio of LD 50 / ED 50 .

[0085] The dosing regimen will be determined by the attending physician and other clinical factors. As is well known in the medical art, the dose for any patient depends on a variety of factors, including the size of the patient, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic checks. The pharmaceutical compositions and formulations described herein are administered at least once to treat or ameliorate or prevent the diseases or disorders described in this specification. However, the pharmaceutical compositions may be administered more than once.

[0086] In other aspects of the present invention, the foregoing compositions are pharmaceutical or cosmetic compositions. In one aspect, the medicament comprising the bioactive neurotoxin can be used for preventing and / or treating at least one of the following diseases or disorders: active myotonia, focal dystonia (including cranial-cervical dystonia and benign essential blepharospasm, hemifacial spasm and focal spasm), gastrointestinal diseases, hyperhidrosis, and cosmetic wrinkle reduction. In another aspect, it also includes blepharospasm, oromandibular dystonia, open-mouth type, closed-mouth type, bruxism, Meige syndrome, lingual dystonia, blepharomuscular disordination, open-mouth type cranial-cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia / adductor type, spasmodic dysphonia / abductor type, spastic dyspnea, limb dystonia, arm dystonia, task-specific dystonia, writer's cramp, musician's finger cramp, golfer's cramp, leg dystonia, thigh adduction, thigh abduction, knee flexion, knee extension, ankle flexion, ankle extension, talipes equinovarus, dystonia of clubfoot, striated toe, toe flexion, toe extension, axial dystonia, Pisa syndrome, belly dancer's dystonia, segmental dystonia, hemidystonia, generalized dystonia, dystonia in Lubag syndrome, dystonia in corticobasal degeneration, dystonia in Lubag syndrome, hypokinetic dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's chorea, dystonia of medullary dysplastic terminal appearance, dopa-induced dyskinesia / dopa-induced dystonia, hypokinetic dyskinesia / hypokinetic dystonia, paroxysmal kinesigenic dyskinesia / dystonia, movement-induced myoclonus of non-motor movements, myoclonic myokymia, rigidity, benign muscle spasm, hereditary chin tremor, paradoxical chin muscle activity, hemimasticatory spasm, hypertrophic masseter myopathy, masseter hypertrophy, anterior tibial muscle hypertrophy, nystagmus, oscillopsia, supranuclear gaze palsy, epilepsy, persistent epilepsy partialis continua, planned spastic torticollis operation, abductor vocal cord paralysis, resistance mutation restlessness, upper esophageal sphincter incompetence, vocal cord granuloma, stuttering Tourette syndrome, middle ear myoclonus, protective laryngeal closure, post-laryngectomy, speech disorder, protective ptosis, entropion, Oddi sphincter dysfunction, pseudoachalasia, non-achalasia esophageal motility disorder, vaginismus, post-operative akinesia tremor, bladder dysfunction, vesical sphincter dysfunction, bladder sphincter spasm, hemifacial spasm, neurogenic kinesigenic dyskinesia, cosmetic use for crow's feet, frowning face asymmetry, mentalis crease, stiff-person syndrome, tetanic prostate hyperplasia, overweight, treatment of strabismus in infantile cerebral palsy, mixed paralytic concomitant strabismus, after retinal detachment surgery, after cataract surgery, myositic strabismus in aphakia, myopathic strabismus,Dissociative vertical deviation, as an adjuvant treatment for strabismus surgery, esotropia, exotropia, akinesia, anal fissure, hypersecretion of exocrine glands, Frey's syndrome, pseudobulbar crying syndrome, hyperhidrosis, axillary palmar plantar rhinorrhea, relative hypersalivation in stroke, relative hypersalivation in Parkinson's disease, relative hypersalivation in the spastic symptoms of amyotrophic lateral sclerosis, relative hypersalivation in encephalitis and myelitis autoimmune diseases, multiple sclerosis, transverse myelitis, Devic's syndrome, viral infection, bacterial infection, parasitic infection, fungal infection, hereditary spastic paraplegia, post-stroke syndrome, cerebral hemisphere infarction, brainstem infarction, spinal infarction, migraine, central nervous system trauma, cerebral hemisphere lesions, brainstem lesions, spinal lesions, central nervous system hemorrhage, intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, spinal cord hemorrhage, in cancer, cerebral hemisphere tumors, brainstem tumors, spinal tumors, snoring (WO 2000 / 033863). For details and symptoms, see, for example, Jost 2007, Drugs 67(5), 669 or Dressler 2000 published in Botulinum Toxin Therapy, Thieme Verlag Publishing Company, Stuttgart, New York.

[0087] In another aspect of the invention, the composition is a cosmetic composition that can be formulated as the above pharmaceutical composition. Similarly for cosmetic compositions, it is contemplated that the compounds of the invention are used in an aspect in substantially pure form. The cosmetic composition is administered intramuscularly in another aspect. In even other aspects of the invention, a cosmetic composition containing a neurotoxin can be formulated as an anti-wrinkle solution.

[0088] In another aspect, the pharmaceutical composition contains an antibody or inhibitor of the invention. Since the polypeptide of the invention is responsible for activating Clostridium neurotoxins, the antibody can be used to reduce the toxic effects observed during Clostridium infections. Thus, the antibody of the invention can be used in one aspect to treat Clostridium infections, including Clostridium perfringens, Clostridium difficile, Clostridium tetani, Clostridium botulinum, Clostridium baratii, Clostridium butyricum, Clostridium sporogenes, Clostridium acetobutylicum, Clostridium haemolyticum, Clostridium novyi, and Clostridium oedematiens. Additionally, the antibody of the invention can be used in another aspect to treat the symptoms associated with the infection. Furthermore, the antibody can be used to treat the conditions or symptoms associated with the disease, where the conditions are selected from botulism, tetanus, pseudomembranous colitis, gangrene, food poisoning, etc.

[0089] In another aspect, the pharmaceutical composition comprises the polypeptide with proteolytic activity of the present invention. The pharmaceutical composition can be used in one aspect for proteolytic cleavage of polypeptides related to co-agglutination, especially for treating patients with insufficient co-agglutination function. In another aspect, the pharmaceutical composition can be used as a fibrinolyticum, especially for treating patients with myocardial infarction, pulmonary embolism, and deep vein thromboembolism, that is, for removing blood clots. It is also contemplated to use the pharmaceutical composition for treating stroke. In addition, in other aspects, the pharmaceutical composition can be used for treating exocrine pancreatic insufficiency to replace trypsin, chymotrypsin, and pepsin. In addition, in other aspects, the pharmaceutical composition can be used for treating patients with inflammatory reactions, for treating cancer patients, especially for proteolytic cleavage of surface-exposed tumor antigens. Additionally, in another aspect, the pharmaceutical composition can be used for treating papilloma.

[0090] The present invention also relates to a method for screening inhibitors, which comprises the following steps: (a) contacting the polypeptide with proteolytic activity of the present invention with a known substrate and an optional putative inhibitor; and (b) determining the effect of the putative inhibitor on the conversion of the substrate into a cleavage product, wherein a decrease in the amount of the cleavage product indicates an inhibitory effect of the putative inhibitor. In one aspect, the putative inhibitor is a peptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 4 to 25, wherein at least one basic amino acid in the amino acid sequence is replaced by a non-basic amino acid. In other aspects, the peptide comprises one or more chemical modifications. In another aspect, the inhibitor is a peptidomimetic of the peptide. In another aspect, the putative inhibitor is part of a chemical compound microarray (i.e., a collection of organic chemical compounds). And in other aspects, the inhibitor is an antibody of the present invention. This method can be used to identify compounds that can inhibit the polypeptide with proteolytic activity of the present invention. The primary screening can be based on, for example, a peptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 4 to 25. Peptides that can inhibit the polypeptide of the present invention can be modified to increase inhibition. The modifications include amino acid substitution or chemical modification. Generally, this method is carried out by contacting the polypeptide of the present invention with a known substrate in the presence and absence of the putative inhibitor (step (a) of this method) and by comparing the effect of the putative inhibitor on the conversion of the substrate into a cleavage product. A decrease in the conversion rate in the presence of the putative inhibitor indicates an inhibitory effect.

[0091] The present invention also relates to an inhibitor of the polypeptide with proteolytic activity of the present invention, wherein the inhibitor is (a) an inhibitor comprising an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 25, wherein the basic amino acid contained therein is replaced by a non-basic amino acid; or (b) an antibody of the present invention.

[0092] All documents cited in this specification are incorporated herein by reference in their entirety, and their disclosures are illustratively mentioned in this specification.

[0093] The figures show:

[0094] Figure 1 : Activity test of fractions collected from HiPrep 16 / 10 Q FF.

[0095] 5 μl of the fraction collected from running HiPrep 16 / 10 Q FF was analyzed for enzyme activity by incubating with 2 μg of scBoNTA (lane 2) at 37 °C for 1 hour and then performing 10% SDS-PAGE. Lane 1: Low molecular weight markers (LMW): 116 kDa, 66 kDa, 45 kDa, 35 kDa.

[0096] Figure 2 : The nBH content (molecular weight about 37.3 kDa) in the fractions collected from SEC (HiLoad 16 / 60 Superdex 75) was analyzed by 12.5% SDS-PAGE. Fractions 9 - 11 mainly contained nBH. (Lane 1: LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa)

[0097] Figure 3 : The purity and protein concentration of three nBH purification batches were determined by 12.5% SDS-PAGE analysis.

[0098] Lane 1, LMW (116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa); Lane 2, nBH lot TE311206 (192 ng / μl mature NT02CB1446 / CBO1444, amino acids 254 - 594 of Genbank accession number CAL82987.1, MW: 38.6 kDa); Lane 3, nBH lot TIK301009 (130 ng / μl mature NT02CB1447 / CBO1445, SEQ ID NO: 1, amino acids 249 - 581 of Genbank accession number CAL82988.1, MW: 37.3 kDa); Lane 4, nBH lot TIK280509 (114 ng / μl mature NT02CB1447 / CBO1445, SEQ ID NO: 1, amino acids 249 - 581 of Genbank accession number CAL82988.1, MW: 37.3 kDa).

[0099] Figure 4 : ESI-MS / MS spectrum analysis report.

[0100] The 38.6 kDa protein band of nBH lot number TE311206 was identified as NT02CB1446 / CBO1444, with a Mascot score of 725 and a peptide MS / MS sequence coverage of 29.6% of the entire open reading frame (ORF). No peptides derived from the first 253 amino acids at the N-terminus were identified (gray boxes identify MS peptides; red squares identify amino acid y- / b-ions of peptides after MS / MS decay). MS / MS analysis of lot number TE311206 showed a sequence coverage of 52% based on the C-terminal amino acids 254 - 594 that form nBH.

[0101] Figure 5 : ESI-MS / MS Spectrum Analysis Report

[0102] The 37.3 kDa protein band of nBH lot number TIK301009 was identified as NT02CB1447 / CBO1445, with a Mascot score of 555 and a peptide MS / MS sequence coverage of 28.4% of the entire open reading frame (ORF). All peptides except one (gray boxes identify MS peptides; red squares identify amino acid y- / b-ions of peptides after MS / MS decay) were identified as derived from the C-terminal 333 amino acids. MS / MS analysis of lot number TIK301009 showed a sequence coverage of 49.5% based on the C-terminal amino acids 249 - 581 that form nBH.

[0103] Figure 6: Comparison of the concentration-dependent proteolytic activities of nBH from three purified batches.

[0104] A The activities of nBH from lot numbers TIK301009, TIK280509, and TE311206 were analyzed by activity test through 12.5% SDS-PAGE, using the following dilutions of concentrated nBH: 1:10, 1:30, 1:100, 1:300, 1:1000. The test was conducted by incubating 1 μg scBoNT / A and 2 μl dH 2 O and 1 μl of the corresponding diluted nBH for 60 minutes at 37 °C. For SDS-PAGE analysis, 3 μl of reducing 4x SDS Laemmli buffer was added to a final volume of 10 μl. 150 kDa scBoNT / A was cleaved into a 100 kDa heavy chain and a 50 kDa light chain.

[0105] B Optical density quantification of the protein bands of the heavy chain, light chain, and scBoNT / A, dividing the sum of the light chain and heavy chain product bands by the sum of the LC, HC, and scBoNT / A protein bands. The first polypeptide at higher dilution factors reduces the cleavage rate. The specific proteolytic activities of the three different batches are almost identical.

[0106] Figure 7 : Time-dependent cleavage of scBoNT / A wild-type and mutants containing modified loops by nBH.

[0107] A loop sequence modification. In scBoNTAS Throm, all lysine residues were removed and the thrombin recognition sequence LVPRGS was inserted instead. In scBoNT Res, the loop lacks any basic amino acids. Shortening the loop to eight small residues or five amino acids with large side chains produced scBoNTAS(GGSG) 2 and scBoNTAS FQWYI. In scBoNTAS CGS-C, the entire loop was deleted and the cysteine-forming disulfide bond was replaced by glycine and serine.

[0108] B SDS-PAGE analysis of time-dependent cleavage of scBoNT / A wild-type and mutants.

[0109] C The scBoNTAS wild-type was activated to the light chain and heavy chain by nBH in a time-dependent manner within 120 minutes. Deletion of lysine and insertion of a single arginine residue prolonged the cleavage of the loop (scBoNTAS Throm). The loop lacking any basic residues was still cleaved (scBoNTAS Res). Shortening the loop to an 8-mer peptide, introducing five amino acids with large side chains, or deleting the entire loop produced non-cleavable scBoNT / A.

[0110] Figure 8: MS / MS analysis of 50 kDa and 100 kDa cleavage products after digestion of scBoNT / A with nBH.

[0111] A Analysis of the 50 kDa cleavage product, which was identified as the light chain of BoNT / A with a Mascot score of 1460. The most C-terminal peptide covered amino acids G433 - K438, which corresponded to the physiologically observed C-terminus of BoNT / A LC.

[0112] B Analysis of the 100 kDa cleavage product, which was identified as the heavy chain of BoNT / A with a Mascot score of 96. The most N-terminal peptide covered amino acids A449 - K456, which corresponded to the physiologically observed N-terminus of BoNT / A HC.

[0113] Figure 9 : A Protein content (mg / ml) of pooled anti-nBH-IgY analyzed by 12.5% SDS-PAGE. B ELISA:Nunc Maxisorp F96 microtiter plates were coated overnight at 4 °C with nBH (500 ng / mL) in different batches of PBS, followed by blocking for 1 h with PBS blocking buffer containing 0.1% Tween-20 and 2% non-fat dry milk. After washing, pooled IgY dilutions (10 μg / ml in blocking buffer) were added for 1 h, followed by detection with biotinylated donkey anti-chicken IgY, streptavidin-horseradish peroxidase (both from Dianova, Hamburg, Germany) and 3,3’,5,5’-tetramethylbenzidine (Sigma).

[0114] Figure 10: A Recombinant expression and isolation of inactive BH 1-581 (63 kDa) by Talon IMAC.

[0115] 10% SDS-PAGE analysis of Talon IMAC fractions (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa; SS34, clear lysate; TD, flow-through; W, wash fraction; E1-E7, imidazole elution fractions 1-7). B No cleavage of scBoNT / A into LC (50 kDa) and HC (100 kDa) (lane 6) by recombinant iBH (SEQ ID NO:2; “E”; 63 kDa) was observed after incubation for 1 h at 37 °C (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa).

[0116] Figure 11: Recombinant purified scBoNT / A of 200 μg of proteolytically processed polypeptide A was obtained using purified active BoNT hydrolase (nBH). 200 μg of recombinant purified scBoNT / A was incubated with 350 ng of purified active BoNT hydrolase at 37 °C for 12 minutes. The reaction was stopped by removing nBH via SEC (column Superdex 200 10 / 300GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / minute), and the amount of cleavage was analyzed by 10% SDS-PAGE. Fraction 1 (1800 μl) containing approximately 40% of the processed BoNT / A was incubated with 350 ng of purified active BoNT hydrolase at 37 °C for 15 minutes and concentrated to 300 μl by ultrafiltration. To finally stop the reaction, nBH was removed via SEC (column Superdex 200 10 / 300GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / minute), and the amount of cleavage was analyzed by 10% SDS-PAGE. Fraction 1 and 2 (1800 μl) containing approximately 80% of the processed BoNT / A were combined and incubated with 120 ng of purified active BoNT hydrolase at 37 °C for 25 minutes and then concentrated to 300 μl by ultrafiltration. To finally stop the reaction, nBH was removed via SEC (column Superdex 200 10 / 300GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / minute), and the amount of cleavage was analyzed by 10% SDS-PAGE. >95% processed BoNT / A (SEQ ID NO.3) was obtained.

[0117] Sequence Listing shows:

[0118] SEQ ID NO: 1: A polypeptide with proteolytic activity derived from Clostridium botulinum strain ATCC 3502, GenBank accession number: "CAL82988.1", lacking 248 N-terminal amino acid residues

[0119] SEQ ID NO: 2: A polypeptide without proteolytic activity derived from Clostridium botulinum strain ATCC 3502, GenBank accession number: "CAL82988.1"

[0120] SEQ ID NO: 3: BoNT / A of ATCC 3502, Genbank accession number "AAA23262"

[0121] SEQ ID NO: 4: The loop body of BoNT / A1

[0122] SEQ ID NO: 5: Loop of BoNT / A2 / A6

[0123] SEQ ID NO: 6: Loop of BoNT / A3

[0124] SEQ ID NO: 7: Loop of BoNT / A3

[0125] SEQ ID NO: 8: Loop of BoNT / A4

[0126] SEQ ID NO: 9: Loop of BoNT / A5

[0127] SEQ ID NO: 10: Loop of BoNT / A7

[0128] SEQ ID NO: 11: Loop of BoNT / B1 / B4bv / B6

[0129] SEQ ID NO: 12: Loop of BoNT / B2 / B3

[0130] SEQ ID NO: 13: Loop of BoNT / B5np

[0131] SEQ ID NO: 14: Loop of BoNT / C / CD

[0132] SEQ ID NO: 15: Loop of BoNT / D

[0133] SEQ ID NO: 16: Loop of BoNT / DC

[0134] SEQ ID NO: 17: Loop of BoNT / E1 - E5

[0135] SEQ ID NO: 18: Loop of BoNT / E6

[0136] SEQ ID NO: 19: Loop of BoNT / F1 / F6

[0137] SEQ ID NO: 20: Loop of BoNT / F2 / F3

[0138] SEQ ID NO: 21: Loop of BoNT / F4

[0139] SEQ ID NO: 22: Loop of BoNT / F5

[0140] SEQ ID NO: 23: Loop of BoNT / F7

[0141] SEQ ID NO: 24: Loop of BoNT / G

[0142] SEQ ID NO: 25: Ring of TeNT

[0143] SEQ ID NO: 26: Nucleic acid sequence encoding SEQ ID NO: 1

[0144] SEQ ID NO: 27: Nucleic acid sequence encoding SEQ ID NO: 2

[0145] The following examples illustrate the invention and should in no case be construed as limiting its scope. Examples

[0146] Example 1: Purification and characterization of native BoNT hydrolase (nBH), which specifically cleaves single-chain BoNT / A into its active double-chain form

[0147] (1) Readout system / activity test : To specifically detect and purify the enzymatic activity that hydrolyzes botulinum neurotoxin A (BoNT / A) into a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) in the culture supernatant of Clostridium botulinum and between chromatographic steps, 150 kDa BoNT / A was expressed as a single-chain (sc) polypeptide in Escherichia coli. Incubation of this recombinant scBoNT / A with the appropriate enzymatic activity (nBH) should produce a 50 kDa LC and a 100 kDa HC that can be observed by reducing 10 - 13% SDS-PAGE.

[0148] (2) Clostripain expression : A single colony of Clostridium botulinum strain ATCC 3502 was inoculated into 100 ml of brain heart infusion (BHI) medium and the culture was incubated overnight at 37°C under anaerobic conditions. 10 ml of the O / N culture was inoculated into 1 l of BHI medium and anaerobically incubated for 48 - 72 hours.

[0149] (3) Ammonium sulfate precipitation : The 1 l culture supernatant was collected by centrifugation (4°C, 6500 x g, 25 minutes). Ammonium sulfate was added to a final concentration of 85% (575 g at this time), the suspension was stirred at 4°C for 6 hours, and then centrifuged (4°C, 6500 x g, 30 minutes). The pelleted ammonium sulfate precipitate was dissolved in a small volume (5 ml at this time) of 50 mM NaP pH 7.5 and dialyzed against 50 mM NaP, 150 mM NaCl pH 7.5. Finally, the dialysate was centrifuged (4°C, 40000 x g, 60 minutes) and the supernatant was used for IEC.

[0150] (4) Ion exchange chromatography (IEC, column HiPrep 16 / 10 Q FF) : The supernatant from (3) Figure 1, lane 3) was applied to a HiPrep 16 / 10 Q FF anion exchange column, which was equilibrated and dialyzed with a buffer containing 50 mM NaP pH 7.5, 150 mM NaCl. The dialysis was carried out at a rate of 1 ml / min. The activity test was performed by incubating 5 μl of all other components and 2 μg of scBoNTA at 37 °C for 1 hour, followed by analysis on SDS-PAGE ( Figure 1 ). Fractions 6 - 24 were combined and concentrated to a volume of 3.5 ml using ultrafiltration (Amicon-Ultra MWCO 10,000).

[0151] (5) Size exclusion chromatography (SEC, HiLoad 16 / 60 Superdex 200) : Subsequently, the concentrated protein solution from (4) was loaded onto a HiLoad 16 / 60 Superdex 200 column, equilibrated with 50 mM NaP pH 7.5, 150 mM NaCl. The separation was carried out at a flow rate of 1 ml / min. Fractions with a retention volume of 80 ml - 100 ml were analyzed using the activity test (1), while the appropriate fractions containing the enzyme activity (nBH) were combined (approx. 10 ml) and concentrated to 3 ml by ultrafiltration. Subsequently, ammonium sulfate was added to a final concentration of 12.5% = 500 mM (+0.2 g).

[0152] (6) Hydrophobic interaction chromatography (HIC, HiTrap phenyl sepharose) : nBH was bound to phenyl sepharose in buffer A (50 mM NaP pH 7.5, 500 mM ammonium sulfate). The bound nBH was eluted by reducing the amount of ammonium sulfate, which was achieved by linearly increasing buffer B (50 mM NaP pH 7.5) at a flow rate of 1 ml / min. All protein-containing fractions were analyzed using the activity test (1), and the appropriate fractions were combined and concentrated to 3.5 ml by ultrafiltration. The buffer of the solution was adjusted to 50 mM NaP pH 7.5; 150 mM NaCl.

[0153] (7) SEC (HiLoad 16 / 60 Superdex 75) : Finally, nBH was purified by SEC using a HiLoad 16 / 60 Superdex 75 column, flow rate 1 ml / min 50 mM NaP pH 7.5, 150 mM NaCl. Fractions with a retention volume of 70 ml - 80 ml were analyzed by 12.5% SDS-PAGE ( Figure 2 ), and the fractions 8 - 12 containing nBH that migrated to approximately 37.3 kDa were combined (approx. 10 ml) and concentrated to 1 ml by ultrafiltration.

[0154] (8) The major protein (nBH) migrating to approximately 37.3 kDa was analyzed by N-terminal peptide sequencing according to the Edman degradation procedure. The identified peptide sequence was V Q G Q S V K G V G and corresponded to the first ten residues of SEQ ID NO: 1.

[0155] (9) Two batches of nBH (NT02CB1447, 37.3 kDa, Figure 3 , lane 3: TIK301009, lane 4: TIK280509) were reproducibly isolated according to the above method. The nBH isoform NT02CB1446 (38.6 kDa, Figure 3 , lane 2, lot number TE311206) was isolated by adjusting the separation method as follows: (i) Growth of Clostridium botulinum culture: 18 hours instead of 48 - 72 hours; (ii) Chromatography step changes: IEC -> SEC Superdex 75 -> HIC phenyl sepharose instead of IEC -> SEC Superdex 200 -> HIC phenyl sepharose -> SEC Superdex 75.

[0156] Example 2: Identification of the sequence of nBH from Clostridium botulinum by mass spectrometry (MS)

[0157] (1) Trypsin digestion : The protein band (nBH) migrating to approximately 38 kDa on SDS-PAGE was excised for trypsin digestion, and it was gently shaken in 50 mM NH 4 HCO 3 , 50% acetonitrile at 37 °C for 30 minutes for decolorization. The decolorization was repeated until the gel spot became transparent. Acetonitrile (100%) was added and removed after 3 minutes. Subsequently, the spot was dried in a vacuum centrifugal drying system (Eppendorf Company, Germany). 50 mM NH 4 HCO 3 containing trypsin (10 ng / μl) was added and incubated on ice for 1 hour. Then, the remaining trypsin solution was removed, a small volume of 50 mM NH 4 HCO 3 was added and digested overnight at 37 °C. The supernatant was collected, and the gel pieces were extracted twice with 5% TFA, 10% acetonitrile. All the liquids were combined, dried in a vacuum centrifugal drying system, and the extracted peptides were stored at 4 °C.

[0158] (2) Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF / TOF) MS: The sample was analyzed in a MALDI-TOF / TOF mass spectrometer (Ultraflex1, Bruker Daltonik GmbH) in linear mode with an acceleration voltage of 25 kV. Masses from 700 m / z to 4,500 m / z were detected. The sample (2 μl) was co-crystallized directly on a stainless steel MALDI target plate with 2 μl of sinapic acid solution containing 50% acetonitrile and 0.2% trifluoroacetic acid (TFA). 500 laser spots were collected for each sample.

[0159] (3) Reverse-phase chromatographic peptide separation : Peptide separation was carried out by reverse-phase chromatography using a nano-HPLC system (Agilent Technologies, Waldbronn, Germany) consisting of an autosampler and a gradient pump. The sample was dissolved in buffer A (5% acetonitrile, 0.1% formic acid), and up to 10 μl aliquots were injected at a flow rate of 5 μl / minute onto a C18 column (Zorbax SB-C18, 5 μm, 300 Å, 0.5 mm inner diameter, 15 cm in length). After loading, the column was washed with buffer A for 15 minutes, and the peptides were eluted with a gradient of eluent A and eluent B (0.1% (v / v) formic acid containing 70% (v / v) acetonitrile) from 0% to 100% eluent B over 75 minutes.

[0160] (4) Electrospray ionization (ESI)-interface and ion trap mass spectrometry : The outlet of the HPLC was directly connected to the nano-ESI source of an ion trap mass spectrometer, and an Agilent coaxial sheath liquid nebulizer (Agilent Technologies) was used. The outlet capillary was fixed by a coiled steel needle, protruding 0.1 - 0.2 mm. The spray was stabilized by N 2 as the nebulizer gas (5 l / minute). The ionization voltage was set at 4,500 V, and the drying gas was supplied at 5 psi and at 250 °C. The spectrometer Esquire3000+ ion trap mass spectrometer (Bruker Daltonik) was used to collect data at a scan rate of 13,000 m / z per second. ESI in positive ion mode was employed, and mass spectrometry data were obtained in the 50 - 1600 m / z scan mode, and could be switched between MS and MS / MS analysis as required by the data. To increase the quality of the MS / MS spectra, only two precursor ions from one spectrum were selected for MS / MS analysis, and the active exclusion was set at 2 minutes to exclude precursor ions that had already been measured.

[0161] (4) Data processing: Data processing was performed using the Data Analysis (version 3.0) and BioTools (version 3.0) software packages (Bruker Daltonik). Protein identification was carried out using the MASCOT software (version 2.1) and the MSDB database (Matrix Science, London, UK).

[0162] (5) Results:

[0163] Table 2: nBH identified by MS

[0164]

[0165] The 38.6 kDa protein band in lane 2 (nBH lot number TE311206) was identified as NT02CB1446 / CBO1444 with a Mascot score of 725 and a peptide MS / MS sequence coverage of 29.6% of the entire open reading frame (ORF). No peptides derived from the first 253 amino acids at the N-terminus were identified ( Figure 4 ). MS / MS analysis of lot number TE311206 showed a sequence coverage of 52% based on the C-terminal amino acids 254 - 594 that form nBH.

[0166] The 37.3 kDa protein bands in lane 3 (nBH lot number TIK301009) and lane 4 (nBH lot number TIK280509) were identified as NT02CB1447 / CBO1445 with Mascot scores of 555 and 609, respectively. All identified peptides, except for one, were derived from the C-terminal 333 amino acids ( Figure 5 ). MS / MS analysis of lot number TIK301009 showed a sequence coverage of 49.5% based on the C-terminal amino acids 249 - 581 that form nBH.

[0167] Example 3: Characterization of nBH enzyme specificity

[0168] (1) Compare the concentration-dependent proteolytic activity of nBH from three purified batches (Figure 6). Analysis of the activity tests of different dilutions of nBH from lots TIK301009, TIK280509, and TE311206 showed that higher dilutions reduced the cleavage rate. The proteolytic activities of these three different lots were almost identical, indicating that the mature isoform NT02CB1446 (TE311206) showed a specific activity similar to that of mature NT02CB1447 (SEQ ID NO: 1).

[0169] (2) Analyze the time-dependent cleavage of nBH on scBoNT / A wild type and mutants using activity tests ( Figure 7)。More than 95% of scBoNTAS wild type was activated into light and heavy chains by nBH in a time-dependent manner within 120 minutes. The loop sequence was modified to characterize the cleavage site. In scBoNTAS Throm, all lysine residues were removed and the thrombin recognition sequence LVPRGS was inserted, which prolonged the cleavage rate. In scBoNT Res, the loop lacked any basic amino acids, which significantly delayed complete hydrolysis, indicating that nBH has a strong recognition preference for basic residues such as lysine and arginine at the cleavage site. In addition, the accessibility of nBH to the loop was hindered by shortening the loop to 8 small residues or 5 amino acids with large side chains (scBoNTAS(GGSG) 2 and scBoNTAS FQWYI).

[0170] (3) MS / MS analysis of the 50 kDa cleavage product after digestion of scBoNT / A with nBH showed that the most C-terminal peptide covered amino acids G433 to K438, which corresponded to the physiologically observed C-terminus of BoNT / A LC( Figure 8A ). Analysis of the 100 kDa cleavage product identified as the heavy chain of BoNT / A demonstrated that the most N-terminal peptide covered amino acids A449 to K456, which corresponded to the physiologically observed N-terminus of BoNT / A HC( Figure 8B ). Thus, the isolated nBH produced processed BoNT / A and preferentially hydrolyzed the peptide bond C-terminal to lysine and arginine residues.

[0171] Example 4: Evolutionary conservation and isotypes of BoNT hydrolases

[0172] Protein sequence analysis of SEQ ID NO: 2 (Genbank accession number CAL82988.1 / YP_001253958.1) showed three conserved domains. Residues 18 - 573 corresponded to zinc metalloprotease (elastase) or LasB involved in amino acid transport and metabolism, with a Blast score of 738. Residues 148 - 212 corresponded to the peptidase propeptide and YPEB domain or PepSY (Blast score 97). Residues 336 - 573 were part of the peptidase M4 family including thermolysin, lysin, aureolysin, and neutral protease (Blast score 803).

[0173] Genome sequencing of Clostridium botulinum ATCC 3502 revealed the presence of six ORFs encoding iBH isotypes (Sebaihia et al., 2007, Genome Res. 17(7):1082-1092). Additional genomic data are available for 10 Group I Clostridium botulinum strains and non-BoNT-secreting spore-forming clostridia, all of which contain 5-7 ORFs encoding iBH. nBH (SEQ ID NO:1) shares at least 64% amino acid sequence identity with 63 other isotypes.

[0174] Example 5: Generation of antibodies specific for BoNT hydrolases

[0175] (1) Production of IgY : Sixteen-week-old chickens [ISA Brown and Lohmann Selected Leghorns (LSL), Spreenhagener Vermehrungsbetrieb für Legehennen GmbH, Bestert, Germany] were housed in individual cages specifically constructed for chickens (Ebeco, Castrop-Rauxel, Germany). Food (ssniff Legehühner-Zucht 1 and 2; Ssniff Co., Ltd., Soest, Germany) and water were available ad libitum. Laying hens started laying eggs at 23 - 25 weeks of age. Eggs were collected daily, marked, and stored at 4 °C until further processing. The housing and experiments of all animals were conducted according to the regulations of the local competent authority in Berlin (No. H0069 / 03). The chickens were immunized and boosted a total of 10 times by intramuscular injection (left and right pectoral muscles) over a 1-year period, with an interval of 4 - 8 weeks between each injection. The intervals used were based on previous studies showing that memory cells did not appear until at least 3 weeks after immunization (Pei and Collisson, 2005). The antigen concentration used was approximately 20 μg (nBH) per injection. No more than 500 μl of the antigen solution was injected per immunization. The first immunization was carried out with complete Freund's adjuvant, and subsequent booster injections were carried out with FIA. The purification of IgY was modified from Polson et al. (1980). Briefly, egg yolks were diluted 1:2 with sterile PBS (pH 7.4, Roche, Mannheim, Germany). To remove fats and lipoproteins, 3.5% (w / v) polyethylene glycol (PEG) 6000 (Roche, Karlsruhe, Germany) was added. After gentle shaking, the mixture was centrifuged (centrifuged at 10,000×g for 20 minutes at 4 °C), the supernatant was poured off, and solid PEG 6000 was added to a final concentration of 12% (w / v). The mixture was then centrifuged as described above. The precipitate was dissolved in 10 ml of PBS, PEG was added to 12% (w / v), and the solution was centrifuged. Finally, the precipitate was dissolved in 1.2 ml of PBS, transferred to a microdialysis device (QuixSep, Roth, Germany), and dialyzed against PBS at 4 °C. The protein content (mg / ml) was analyzed by 12.5% SDS-PAGE ( Figure 9 A) and detected with a photometer at 280 nm. The extinction coefficient of IgY was calculated to be 1.33 according to the Lambert-Beer law.

[0176] (2) ELISA : Nunc Maxisorp F96 microtiter plates (VWR International GmbH, Darmstadt, Germany) were coated overnight at 4 °C with PBS containing different batches of nBH (500 ng / mL), and then blocked for 1 hour with a solution containing 0.1% Tween-20 and 2% non-fat dry milk (Merck, Darmstadt, Germany). After washing, IgY diluent (10 μg / ml in blocking buffer) was added for 1 hour, followed by detection using biotinylated donkey anti-chicken IgY, streptavidin-horseradish peroxidase (both from Dianova, Hamburg, Germany), and 3,3’,5,5’-tetramethylbenzidine (Sigma). The detected nBH was as Figure 9 shown in B.

[0177] (3) Western blot: nBH was separated by 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Karlsruhe, Germany) using standard immunoblotting techniques. The membrane was blocked overnight at 4 °C and incubated with IgY (1:5,000 in blocking buffer) for 1 hour. After washing, the membrane was probed with biotinylated donkey anti-chicken IgY for 30 minutes and developed using alkaline phosphatase and CDP-Star (Perkin Elmer, Waltham, MA).

[0178] Example 6: Recombinant expression of BoNT hydrolase

[0179] (1) Plasmid construct : The gene portions encoding native BH (SEQ ID NO: 1) and its propeptide (SEQ ID NO: 2) were amplified by PCR using appropriate oligonucleotides and genomic DNA of Clostridium botulinum ATCC 3502, which were fused with oligonucleotides encoding His6-tag and inserted into pQE3 (Qiagen) to generate expression plasmids pQ-BH1445H6-249-581 and pQ-BH1445H6-1-581, respectively. The nucleotide sequences were verified by DNA sequencing.

[0180] (2) Purification of recombinant protein : nBH and iBH fused with a carboxyl-terminal His6-tag were generated by incubating with Escherichia coli strain M15pREP4 (Qiagen) at room temperature for 10 hours and purified using Talon-agarose beads (Clontech) according to the manufacturer's instructions. The fractions containing the desired proteins were pooled, frozen in liquid nitrogen, and stored at -70 °C. iBH was isolated as a recombinant protein with an MW of 63 kDa ( Figure 10A ). The inactivity of iBH was shown by activity assays, and after incubation at 37 °C for 1 hour, no scBoNT / A wild type (wt) was hydrolyzed into LC and HC ( Figure 10B ).

[0181] Example 7: Inhibition of BoNT hydrolase

[0182] (1) Screening for peptide inhibitors of BH: Peptides based on SEQ ID NOs: 4 - 25 lacking one or more basic residues were synthesized. Each peptide was added to the mixture according to the activity assay. Peptides that could reduce the amount of treated scBoNT / A, prolong the time required for complete treatment of scBoNT / A, or block the treatment of scBoNT / A were considered inhibitors of nBH.

[0183] (2) Screening of antibody-based inhibitors: Antibodies generated against epitopes derived from nBH, such as the IgY of Example 5, were incubated with nBH, followed by activity testing. Antibodies that could reduce the amount of processed scBoNT / A, prolong the time required for complete processing of scBoNT / A, or block the processing of scBoNT / A were regarded as inhibitors of nBH.

[0184] Example 8: Obtaining proteolytically processed polypeptides using purified active BoNT hydrolase (nBH)

[0185] (1) 200 μg of recombinant purified scBoNT / A was incubated with 350 ng of purified active BoNT hydrolase at 37 °C for 12 minutes. To stop the reaction, nBH was removed by SEC (column Superdex 200 10 / 300GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / minute), and the amount of cleavage was analyzed by 10% SDS-PAGE ( Figure 11A ).

[0186] (2) Fraction 1 (1800 μl) containing approximately 40% of processed BoNT / A was incubated with 350 ng of purified active BoNT hydrolase at 37 °C for 15 minutes and concentrated to 300 μl by ultrafiltration. Finally, to stop the reaction, nBH was removed by SEC (column Superdex 200 10 / 300GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / minute), and the amount of cleavage was analyzed by 10% SDS-PAGE ( Figure 11B )

[0187] (2) Fractions 1 and 2 (1800 μl) containing approximately 80% of processed BoNT / A were combined and incubated with 120 ng of purified active BoNT hydrolase at 37 °C for 25 minutes and concentrated to 300 μl by ultrafiltration. Finally, to stop the reaction, nBH was removed by SEC (column Superdex 200 10 / 300GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / minute), and the amount of cleavage was analyzed by 10% SDS-PAGE ( Figure 11C)。Obtain >95% of the processed BoNT / A (Seq ID NO.3). If the second polypeptide is processed for 50 minutes in one step at 37 °C (200 μg scBoNT / A incubated with 350 ng nBH), the same fully processed second polypeptide (>95% processed BoNT / A) is obtained. After incubation at 37 °C for 1 hour, more than 97% of the BoNT / A is processed.

Claims

1. A polypeptide having proteolytic activity, which comprises a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:

1.

2. A nucleic acid molecule, which comprises a nucleic acid sequence encoding the polypeptide of claim 1 and optionally regulatory elements.

3. A vector, which comprises the nucleic acid molecule as described in claim 2.

4. A cell, which comprises the nucleic acid molecule as described in claim 2 or the vector as described in claim 3.

5. A method for preparing a polypeptide having proteolytic activity, the method comprising the following steps: (a.) Chemically synthesizing or translating a polypeptide from a nucleotide sequence, the polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:1; and (b.) Purifying the polypeptide in step (a.).

6. A polypeptide obtainable by the method as described in claim 5.

7. An antibody, which specifically binds to the polypeptide as described in claim 1 or 6.

8. Use of the antibody as described in claim 7 in a method for purifying the polypeptide as described in claim 1 or 6.

9. A method for preparing a proteolytically processed polypeptide, the method comprising the step of contacting the following substances (a) with (b): (a) A first polypeptide, which is selected from the polypeptide as described in claim 1 or 6, Lys-N, Lys-C, arginyl endopeptidase, plasmin, or Omptin protein, (b) A second polypeptide, which is susceptible to proteolysis by the first polypeptide; wherein the contacting results in the proteolytic processing of the second polypeptide into at least two cleavage products.

10. The method as described in claim 9, characterized in that, the second polypeptide comprises an amino acid sequence having at least 50% sequence identity with a polypeptide sequence selected from any one of SEQ ID NO:3 to 25; preferably, wherein preferably the first polypeptide proteolytically cleaves the second polypeptide at the direct C-terminal position of a basic amino acid residue (e.g., His, Lys, Arg) in any one of the sequences of SEQ ID NO:3 to 25.

11. The method as described in claim 9 or claim 10, characterized in that, the second polypeptide is a clostridial neurotoxin (e.g., BoNT / A).

12. The method as described in claim 11, characterized in that, The Clostridial neurotoxin is selected from: a Clostridial neurotoxin polypeptide lacking a functional binding domain (H CC ) and thus unable to bind to a native Clostridial neurotoxin receptor (e.g., a polypeptide comprising or consisting of the LH N fragment of a Clostridial neurotoxin), a Clostridial neurotoxin polypeptide having a modified Clostridial neurotoxin binding domain (H CC ) that binds to a native Clostridial neurotoxin receptor, or a Clostridial neurotoxin polypeptide having a non-Clostridial binding domain that causes the Clostridial neurotoxin to bind to a non-native Clostridial neurotoxin receptor (the polypeptide optionally lacking a functional binding domain (H CC ) to minimize binding of the Clostridial neurotoxin to a native Clostridial neurotoxin receptor).

13. The method as described in claim 12, characterized in that, the L-chain and H-chain components of the clostridial neurotoxin are from the same or different clostridial neurotoxin serotypes and / or subtypes.

14. The method as described in any one of claims 9-13, characterized in that, the second polypeptide is a single-chain clostridial neurotoxin prepared by recombinant expression in Escherichia coli.

15. The method as described in any one of claims 9-14, characterized in that, the second polypeptide is a single-chain clostridial neurotoxin, and wherein the contacting of the first and second polypeptides produces a clostridial neurotoxin double-chain polypeptide, the clostridial neurotoxin double-chain polypeptide comprising a clostridial neurotoxin L-chain component covalently linked to the clostridial neurotoxin H-chain component via a disulfide bond.

16. The method as described in any one of claims 9-15, It is characterized in that the proteolytically treated second polypeptide is a Clostridium neurotoxin double-stranded polypeptide, wherein the C-terminus of the L-chain and the N-terminus of the H-chain are the same as the corresponding termini of the corresponding double-stranded Clostridium neurotoxin generated from the same single-stranded Clostridium neurotoxin polypeptide in wild-type Clostridium.

17. The method according to any one of claims 9-16, It is characterized in that the proteolytically treated second polypeptide is a Clostridium neurotoxin double-stranded polypeptide, which has the same amino acid sequence as the corresponding Clostridium neurotoxin double-stranded polypeptide generated from the same single-stranded Clostridium neurotoxin polypeptide in wild-type Clostridium.

18. Use of the method according to any one of claims 9-17 in product quality assessment or drug preparation.

19. A composition obtainable by the method according to any one of claims 9-18, It is characterized in that the composition comprises a mixture of treated and untreated second polypeptides, and the mixture comprises less than 5% of the untreated second polypeptide.

20. A method for screening an inhibitor, the method comprising the following steps: (a) contacting the polypeptide according to claim 1 or 6 with a known substrate and an optional putative inhibitor; and (b) detecting the effect of the putative inhibitor on the conversion of the substrate into a cleavage product wherein, a decrease in the amount of the cleavage product indicates the inhibitory effect of the putative inhibitor.

21. An inhibitor of the proteolytically active polypeptide according to claim 1 or 6, It is characterized in that the inhibitor is (a.) an inhibitor comprising an amino acid sequence shown in any one of SEQ ID NO: 4-25, wherein the basic amino acids comprised are replaced by non-basic amino acids; or (b.) an antibody according to claim 7.

22. A pharmaceutical composition comprising the polypeptide according to claim 1 or 6, the antibody according to claim 7, the composition according to claim 19 or the inhibitor according to claim 21.

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