RU2719164C1 - Methods for producing proteolytically processed polypeptides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to the use of Lysylendopeptidase Lys-C from Lysobacter enzymogenes in the processing of botulinum neurotoxin of serotype F (BoNT/F).

EFFECT: use of Lys-C for proteolytic splitting of single-stranded BoNT/F enables to obtain an active double-stranded BoNT/F.

9 cl, 11 dwg, 2 tbl, 8 ex

Description

The present invention relates to a new proteolytically active polypeptide and various uses of the polypeptide in screening and production methods.

Clostridium botulinum and Clostridium tetani produce potent neurotoxins, i.e. botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT), respectively. These clostridial neurotoxins (CNTs) specifically bind to nerve cells and disrupt the release of neurotransmitters. Clostridium botulinum secretes seven antigenic variants of serotypes of botulinum neurotoxin (BoNT), denoted by the letters A to G. All serotypes, together with the related tetanus neurotoxin (TeNT) secreted by Clostridium tetani , are Zn ²⁺ endoproteases that block synaptic exocytosis, involved in the formation of the complex SNARE, which controls the fusion of cell membranes. CNTs cause peripheral muscle paralysis seen in botulism and tetanus. It was shown that the activity of CNT affects the secretion of glands. These physiological effects of CNT on muscle and gland activity are increasingly used for various therapeutic and cosmetic purposes. Botulinum neurotoxin serotype A (BoNT / A) was approved for use in humans in the United States in 1989 for the treatment of strabismus, blepharospasm and other disorders. It is commercially available as a protein preparation of botulinum neurotoxin A, for example, under the trade name BOTOX (Allergan Inc.) and under the trade name DYSPORT (Ipsen Ltd.). In therapeutic use, a complex comprising neurotoxin and additional bacterial proteins is injected directly 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 the desired pharmacological effect develops. An improved BoNT / A preparation without complexing proteins is available under the trade names XEOMIN or Bocouture (Merz Pharmaceuticals GmbH, Frankfurt / Germany). The BoNT effect is only temporary, which is the reason for the repeated administration of BoNT, which is usually required to maintain a therapeutic effect.

Each CNT is initially synthesized as an inactive single chain polypeptide. In the case of BoNT, the neurotoxin polypeptide has a molecular weight of approximately 150 kDa. The post-translational processing of this single-stranded polypeptide involves limited proteolysis of the protruding region called the loop (see Table 1) and the formation of a disulfide bridge nearby. An active double-stranded neurotoxin consists of two cleavage products resulting from proteolytic hydrolysis of a single-stranded precursor polypeptide: an N-terminal light chain of about 50 kDa and a heavy chain of about 100 kDa linked by a disulfide bond. The CNT structure consists of three domains, i.e. a catalytic light chain, a heavy chain comprising a translocation domain (N-terminal half) and a receptor binding domain (C-terminal half) (cf. 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 in one chain between amino acid residues forming a catalytic domain and amino acid residues forming a translocation domain, endopeptidase activity can lead to the formation of two large cleavage products, i.e. light and heavy chains, and, in addition, characteristic short peptides representing the former region of the loop connecting in a single-stranded neurotoxin sections that will become light and heavy chains (cf. Table 1, below).

The purification of CNT from a fermentation solution is particularly difficult since it contains neurotoxins as a mixture of unprocessed, partially processed and fully processed polypeptides that have similar biochemical and physical properties. Typically, partially processed neurotoxins are formed 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. Moreover, a partially processed neurotoxin can also form if endoproteolytic activity has led to the separation of 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 yet been hydrolyzed. Depending on the fermentation conditions and the type of neurotoxin, a fully processed polypeptide lacking a loop peptide may be significantly contaminated from 5% to 90% with partially processed or unprocessed peptides. However, in some cases, the neurotoxin is mainly unprocessed and needs to be treated with endopeptidase before it is used for therapy to become biologically active.

The prior art describes various attempts to treat clostridial neurotoxins with heterologous proteases in order to reduce the number of unprocessed or partially processed precursor proteins. The protease most commonly used to activate clostridial neurotoxins, trypsin, is useful for activating clostridial neurotoxins of serotypes B (BoNT / B) and E (BoNT / E) (DasGupta & Sugiyama 1972, Biochem. Biophys. Res. Commun. 48: 108- 108. 112; Kozaki et al ., 1974, Infect. Immun. 10: 750-756) probably forms secondary products, possibly through a proteolytic action near the C-terminus of the heavy BoNT / A subunit, and thus likely disrupts toxin binding with its cellular receptor (Shone et al ., 1985, Eur. J. Bioch. 151: 75-82). The formation of more specific cleavage products is theoretically expected from endogenous proteases isolated from a natural host, such as C. botulinum , forming BoNT / A. Accordingly, various attempts are made to isolate endogenous proteases involved in the proteolytic activation of clostridial neurotoxins from a natural host cell. Dekleva and DasGupta (Dekleva & DasGupta, 1989, Biochem. Biophys. Res. Commun. 162: 767-772) were purified from C. botulinum cultures forming BoNT / A, a fraction capable of proteolytically cleaving BoNT / A into heavy and light subunits. Later studies by the same authors further characterized the endogenous protease isolated from C. botulinum (Dekleva & DasGupta 1990, J. Bact. 172: 2498-2503) and revealed 62 kDa protein consisting of 15.5 kDa polypeptide and 48 kDa polypeptide. However, the observation of significant CNT fragmentation after the limited exposure to 62 kDa protein detected by Dekleva and DasGupta suggests that the isolated protease may not be the unidentified proteolytic enzyme responsible for CNT activation in clostridia cell cultures and during infection. In fact, other authors have recently suggested that clostripain, also called clostridiopeptidase B (Mitchel & Harrington, 1968, JBC 243: 4683-4692), may be involved in specific activation of CNT (Sebaihia et al., 2007, Genome Res. 17 (7 ): 1082-1092; WO2009 / 014854). Interestingly, the structure and substrate specificity of this enzyme resemble those of secreted alpha-clostripain from Clostridium histolyticum (Dargatz et al. 1993), whose homologue (74% amino acid identity) is present in C. botulinum (CBO1920). Alpha-clostripain C. histolyticum is a cysteine endopeptidase with strict specificity for arginine bonds. It is synthesized as an inactive preproenzyme that undergoes autocatalytic cleavage to form 15.4 and 43 kDa polypeptides that combine to form a heterodimeric active enzyme (Dargatz et al. 1993). Both C. histolyticum alpha-clostripain and 62 kDa protease of C. botulinum require a reducing agent and calcium to be fully active and are subject to the same protease inhibitors. These data strongly suggest that alpha-clostripain ortholog of C. botulinum (CBO1920) is an endogenous protease responsible for the proteolytic cut neurotoxin C. botulinum. The gene encoding clostripain (CPE0846) is also present in C. perfringens and has been shown to be positively regulated by the two-component VirR / VirS system (Shimizu et al. 2002b).

To date, however, further convincing evidence is lacking, and a protease capable of effectively converting a single-stranded CNT precursor into true mature cleavage products, i.e. double-stranded neurotoxin is still not available in the art. The present invention solves one or more of the above problems.

Means and methods of reducing the number of unprocessed and / or partially processed neurotoxin polypeptides and thus improving the quality of neurotoxin preparations are in demand, but still inaccessible. Thus, the technical problem underlying the present invention can be considered as a representation of means and methods for improving the production of neurotoxin polypeptides by satisfying the requirements described above. The technical problem is solved by means of the application of the invention described in the claims and below.

Accordingly, the present invention relates in one aspect to a proteolytically active polypeptide, which comprises 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 proteolytically active polypeptide consisting 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 proteolite cally active polypeptide consisting of the polypeptide sequence as shown in SEQ ID NO: 1.

The term "proteolytically active polypeptide" in this context refers to the catalytic function of the polypeptide of the invention, and means that the polypeptide of the invention is capable of hydrolyzing the peptide bond. In one aspect, a “proteolytically active polypeptide” refers to a polypeptide that is capable of hydrolyzing a polypeptide comprising an amino acid sequence selected from any of SEQ ID NOs: 4 to 25. The term “proteolytically inactive polypeptide” in this context refers to the catalytic function of a polypeptide that is the subject of the present invention, and means that the polypeptide that is the subject of the present invention is not able to hydrolyze the peptide bond.

One of skill in the art can determine whether a polypeptide, in accordance with the definition of sequence mentioned herein, is a polypeptide in accordance with the present invention, by testing the proteolytic activity of the above polypeptide. A study or test system for determining proteolytic activity includes bringing the polypeptide, which includes a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1, in contact with the test substrate. The test substrate is usually a polypeptide, which is known to be cleaved by the polypeptide of the invention. Preferably, the test substrate is CNT, such as BoNT, or a fragment thereof. The test substrate may be, for example, uncleaved / unprocessed BoNT, denoted scBoNT in the present description, and may be, for example, serotypes A, B, C1, D, E, F or G (for example, “scBoNT / A”, “scBoNT (B ", etc.), or the test substrate may be a tetanus neurotoxin. Alternatively, the test substrate may be a clostridial neurotoxin fragment, the aforementioned fragment comprising an amino acid sequence selected from any of SEQ ID NOs: 4 to 25. The fragment may be a polypeptide of 50 or more amino acid residues or a peptide of up to 49 amino acid residues. As used throughout this technical description, the term "polypeptide" refers to a molecule of 50 or more amino acid residues, while the term "peptide" refers to a molecule of 2-49 amino acid residues. In one aspect, the test substrate is a soluble fragment of a neurotoxin called LH _N , comprising a light chain polypeptide, a protruding region of a loop peptide and an N-terminal half of the heavy chain polypeptide, an H _N translocation domain. In another aspect, the test substrate is a peptide or comprises a peptide selected from any of SEQ ID NO: 4 to 25 (cf. Table 1). In yet another aspect, the test substrate is a chimeric neurotoxin comprising amino acid residues derived from two or more serotypes.

A study to determine proteolytic activity will usually include the step of determining the level of conversion of the test substrate to its cleavage product (s). The observation of one or more cleavage products formed after contact of the polypeptide with the test substrate, or the observation of an increase in the amount of cleavage product (s), is an indicator of the proteolytic activity of the polypeptide. The above determination step may include comparing the substrate with the cleavage product (s). The above comparison may include determining the amount of substrate and / or the amount of one or more cleavage products, and may also include calculating the ratio of substrate and cleavage products. In addition, a study to determine proteolytic activity may include the step of comparing the test sample with the reference sample, in which the reference sample typically includes (a) a polypeptide that comprises a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1, and which is known to have proteolytic activity, and (b) a test substrate, which is known to be cleaved by the polypeptide according to (a). In one aspect, a study to determine proteolytic activity includes separating the substrate and the cleavage product (s) by electrophoresis or column chromatography and, if necessary, spectrometric analysis. It may be convenient to label the test substrate with one or more tags to facilitate detection of a decrease in the amount of test substrate and / or an increase in the amount of product (s). The term "label" in this context refers to a detectable marker and includes, for example, a radioactive label, an antibody, a fluorescent label. The amount of test substrate and / or decay product can be determined, for example, by autoradiography or spectrometry methods, including methods based on the transfer of resonant energy between at least two labels. Alternatively, immunological methods such as Western blot or enzyme-linked immunosorbent assay (ELISA) can be used for detection. A preferred study for determining the proteolytic activity of a polypeptide of the invention is described below in the examples illustrating the invention. In a particularly preferred embodiment of the present invention, the polypeptide is proteolytically active if more than 20%, preferably more than 95% of the test substrate is converted to cleavage products, such as light chains and heavy chains, in 120 minutes at 37 ° C using a buffer selected from 100 mM Tris-HCl, pH 8.0 or phosphate-saline solution (50 mM Na ₂ PO ₄ , 150 mM NaCl, pH 7.4). The same conditions apply if the test substrate is not a full-sized neurotoxin, but, for example, a fragment of a full-sized neurotoxin or a derivative of a neurotoxin. Obviously, in this case, the cleavage products will be different. However, one skilled in the art can determine the amount of the corresponding cleavage products. In another aspect, 100 ng of a proteolytically active polypeptide and a molar ratio of 1: 100 relative to the substrate are typically used in the study. In yet another aspect, a sample can be sampled at certain intervals to monitor catalytic activity over time. The study can be modified, for example, using different amounts of proteolytically active polypeptide.

SEQ ID NO: 2 shows the polypeptide sequence of a proteolytically inactive polypeptide obtained from Clostridium botulinum strain ATCC 3502, GenBank number “CAL82988.1”, the length of which is 581 amino acid residues. SEQ ID NO: 1 shows a proteolytically active derivative of SEQ ID NO: 2 lacking amino acid residues at positions 1 through 248 of the sequence SEQ ID NO: 2.

The term "polypeptide that includes a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1" refers to a polypeptide that has at least 50% sequence identity with 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 with the sequence of SEQ ID NO: 1. The above polypeptide may contain additional amino acids, for example, in the internal position or at the N- or C-terminus in the sequence shown in SEQ ID NO: 1, or in the internal position or at the N- or C-terminus of a sequence having at least 50% identity with the sequence of SEQ ID NO: 1, in which methionine may be present at the N-terminus of the polypeptide. In addition, the term refers to a polypeptide in which one or more amino acid residues are absent, for example, in the internal position or at the N- or C-terminus of the sequence shown in SEQ ID NO: 1, or in the internal position or at N- or C the end of a sequence having at least 50% identity with the sequence of SEQ ID NO: 1.

The term "sequence identity" in this context refers to the definition of identity between the amino acid sequence of comparison and the analyzed sequence, in which the sequence is aligned in such a way as to obtain the maximum match, and which can be calculated using techniques or methods encoded in computer programs, such as e.g. BLASTP, BLASTN, FASTA (Altschul 1990, J Mol Biol 215: 403). Percent identity values in one aspect are calculated for the entire amino acid sequence. In another aspect, sequence identity is calculated for a sequence length of up to 50 a.k. (amino acid) residues, up to 100 a.k., up to 150 a.k., up to 250 a.k., 300 a.k., 350 a.k., 400 a.k., 450 a.k., 500 a.k. or 550 a.k. residues. In another aspect, sequence identity is calculated for at least 50 a.k. residues of at least 100 a.k., at least 150 a.k. or at least 250 a.k. residues. In preferred embodiments of the invention, sequence identity is determined for the entire length of SEQ ID NO: 1 or 2, i.e. for length 333 a.k. or 581 a.k., respectively. A series of programs based on different algorithms is available to specialists for comparing different sequences. In this context, the Needleman and Wunsh or Smith and Waterman algorithms give particularly reliable results. For alignment of sequences and calculation of sequence identity values listed in the present description, we used the commercially available DNASTAR Lasergene MegAlign software version 7.1.0 based on the Clustal W algorithm for the entire sequence area with the following settings: pair alignment parameters: penalty for skipping a sequence: 10.00; fine for elongation of the gap: 0.10; a Gonnet 250 amino acid comparison matrix, which, unless otherwise specified, should always be used as standard settings for sequence alignment.

The term "at least 50% sequence identity" in this context means 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%.

The proteolytically active polypeptide of the invention may contain the same amount of amino acids as the comparison polypeptide sequence as shown in SEQ ID NO: 1. Also included in the present invention are polypeptides having additional amino acid residues or having fewer amino acid residues. In one aspect, the proteolytically active polypeptide of the present invention is or comprises a truncated mutant of SEQ ID NO: 1 or 2 or a polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 1 or 2. The truncated mutant SEQ ID NO: 2, for example, there may not be one or more amino acid residues located at the N-terminus of the amino acid at position 249. The truncated mutant may be an N- or C-terminal truncated mutant and / or an internal truncated mutant that is proteolytically active. In one aspect, the aforementioned truncated mutant of SEQ ID NO: 2 lacks amino acid positions 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 aforementioned truncated mutant has no SEQ ID NO: 2 sequence 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 length of the proteolytically active polypeptide of the invention is at least 200 a.k. residues of at least 250 a.k. residues of at least 300 a.k. residues or at least 333 a.k. the remainder. In another aspect, the proteolytically active polypeptide of the invention comprises up to 333 a.k. residues, up to 350 a.k. residues, up to 573 a.k. residues, up to 592 a.k. residues, up to 600 a.k. residues or up to 617 a.k. residues.

In another aspect, the proteolytically active polypeptide of the invention comprises a polypeptide comprising additional amino acid residues at the N- or C-terminus and / or in the internal position of the polypeptide chain of SEQ ID NO: 1 or a polypeptide sequence having at least 50% identity sequences with the sequence of SEQ ID NO: 1. These additional amino acid residues may include up to 5, up to 10, or even up to 200, 300 or up to 400 consecutive amino acid residues. In one aspect, additional amino acid residues act as an inhibitor of proteolytic activity. In another aspect, additional amino acid residues can be removed with a protease. In another aspect, additional residues inhibiting the proteolytic activity of the polypeptide of the invention are removed. Additional amino acid residues may be flanked by one or more protease cleavage sites. In another aspect, the additional amino acid sequence acts as a detectable marker and / or provides binding to a solid support.

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 replacing one or more amino acid residues. The term "replacement" in this context means the replacement of an amino acid with another amino acid. For example, up to 1 a.k., 2 a.k., 3 a.k., 4 a.k., 5 a.k., 6 a.k., 7 a.k., 8 a.k., 9 a.k., 10 a.k., 15 a.k., 20 a.k. or up to 50 a.k. can be replaced within the polypeptide sequence. Substitutions may include conservative or non-conservative amino acid substitutions made to, for example, increase or decrease the binding of the substrate or the proteolytic activity of the polypeptide of the invention.

In one aspect, a proteolytically active polypeptide of the invention includes a polypeptide that is capable of hydrolyzing a substrate to two or more native cleavage products. In another aspect, the polypeptide of the present invention hydrolyzes the substrate to two or more cleavage products that are different from native cleavage products. The term "native cleavage products" or "native products" in this context refers to products that are identical in amino acid sequence when compared with products formed from the same substrate in the wild-type cultures from which the substrate is derived. In one aspect, the cleavage product is a double-stranded neurotoxin of a botulinum neurotoxin or tetanus neurotoxin, in another aspect, a double-stranded neurotoxin is a neurotoxin isolated from C. botulinum serotype A, B, C1, D, E, F or G. In another aspect, the above double-stranded neurotoxin is native double-stranded neurotoxin.

Table 1 shows the precursor, the native double-stranded neurotoxin TeNT and BoNT / A-G, and identifies a protruding loop including the amino acid sequence cleaved by the polypeptide of the present invention

Table 1

It should be understood that the definitions and explanations of the terms above and below apply, subject to the necessary changes, to all aspects described in this description of the invention, unless otherwise specified.

The proteolytically active polypeptide of the invention is suitable for various applications. A commercially relevant application is their use in the manufacture of therapeutic neurotoxins, such as those derived from C. botulinum . Currently, C. botulinum cell cultures used to produce commercially available botulinum neurotoxin preparations are contaminated with a significant amount of partially processed and / or unprocessed neurotoxin, which have a negative effect, i.e. reduce the specific activity of these pharmaceutical compositions. Using the proteolytically active or activated polypeptide of the present invention, for example, after lysis of C. botulinum , it will be possible to process compositions comprising an unprocessed and / or partially processed neurotoxin, and thus convert these contaminants into a fully processed neurotoxin. As a result, commercial products with increased specific neurotoxin activity can be made in which the total amount of bacterial protein can be reduced, further reducing the risk of antibody formation in the patient.

In another aspect, the present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide of the invention and, in some cases, regulatory elements. The term "regulatory elements" as used herein refers to regulatory elements of gene expression, including transcription and translation, and includes elements such as a TATA box, promoter, enhancer, ribosome binding site, Shain-Dalgarno sequence, ribosome internal landing site (IRES) , polyadenylation signal, end capping structure and the like. The above regulatory element may include one or more heterologous regulatory elements or one or more homologous regulatory elements. A “homologous regulatory element” is a regulatory element of a wild-type cell from which the nucleic acid molecule of the present invention is obtained, which is involved in regulating the expression of a gene of a nucleic acid molecule or polypeptide in the aforementioned wild-type cell. The present invention also includes nucleic acid molecules comprising heterologous regulatory elements. The term “heterologous regulatory element” is a regulatory element that is not involved in the regulation of gene expression of a nucleic acid molecule or polypeptide in the above wild-type cell. Also included are regulatory elements for inducible expression, such as inducible promoters. The nucleic acid molecule may be, for example, hyRNA, mRNA, RNA, DNA, PNA, LNA and / or modified nucleic acid molecules. The nucleic acid molecule may be cyclic, linear, integrated into the genome or episomal. Also included are concatemers encoding fusion proteins comprising three, four, five, six, seven, eight, nine or ten polypeptides that are the subject of the present invention. Moreover, the nucleic acid molecule may contain sequences encoding signal sequences for intracellular transport, such as signals for transport to the intracellular compartment or for transport across the cell membrane.

In another aspect, the present invention relates to a vector comprising a nucleic acid molecule, in accordance with the nucleic acid molecule that is the subject of the present invention. The vector may be suitable for in vitro and / or in vivo expression of the polypeptide of the invention. The vector may be a vector for transient and / or stable gene expression. In one application of the present invention, the vector further comprises regulatory elements and / or selective markers. The above vector in one application of the present invention has a viral origin, in another application of the present invention has a phage origin, in another embodiment of the present invention has a bacterial origin.

In another aspect, the present invention relates to a cell comprising a nucleic acid molecule or vector that is the subject of the present invention. The term "cell" in this context includes prokaryotic and / or eukaryotic cells suitable for expression of the aforementioned nucleic acid molecule or the aforementioned vector, and in particular the polypeptide of the invention. The aforementioned cell may be a host cell that does not express the polypeptide of the invention, or a homolog thereof. The term “homologue” as used herein refers to a polypeptide comprising a polypeptide sequence having a 50% sequence identity with the sequence of SEQ ID NO: 1. However, cells, in particular wild-type cells expressing the polypeptide of the present invention, are also included in the present invention, or his homologue. In a particular aspect, the cell of the invention is selected from C. botulinum, C. butyricum, C. baratii and C. tetani. In a preferred aspect, the cell is C. botulinum serotype A, B, or F. In another aspect, the cell is Hall strain (ATCC 3502) C. botulinum . In another aspect, the cell is a BoNT / A-forming strain of ATCC 19397, also known as NCTC 4587 and NCTC 7272 C. botulinum . In another aspect, the aforementioned cell is a BoNT / A-forming strain of NCTC 2916 C. botulinum . In another aspect, the above cells are BoNT / A2-forming strains of Kyoto-F and Mauritius / NCTC 9837 C. botulinum . In another aspect, the aforementioned cell is a BoNT / A3 forming strain A254 of Loch Maree / NCTC 2012 C. botulinum . In another aspect, the aforementioned cell is a BoNT / A4 and B strain C. botulinum CDC657. In another aspect, the aforementioned cell is BoNT / A5 and B3 'gene forming C. botulinum strain H04402 065. In another aspect, the aforementioned cell is a BoNT / B1-forming strain of Okra / NCTC 7273 C. botulinum . In another aspect, the above cell is BoNT / B and F strain C. botulinum forming CDC4013 / NCTC 12265. In another aspect, the aforementioned cell is a BoNT / F1-forming strain of C. botulinum Langeland / NCTC 10281. In another aspect, the above 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 yeast cell. In one aspect, the polypeptide of the present invention is modified within the cell (i.e., glycosylated, phosphorylated, processed by proteases, etc.). Modification also includes the addition of non-protein cofactors, including metal ions. Cells comprising the proteolytically inactive polypeptide described above, any intermediate polypeptide product, as well as the final proteolytically active polypeptide disclosed herein, are included in the present invention. Also included in the present invention are cells comprising an expression inducer of a polypeptide of the invention. Such an expression inducer may be a nucleic acid molecule, a polypeptide or a chemical structural element, including small chemical structural elements, having the effect of increasing the amount or activity of the proteolytically active polypeptide of the invention in cell cultures or their lysates. An expression inducer may, for example, increase the transcription or translation of a nucleic acid molecule encoding a polypeptide of the invention. Alternatively, the expression inducer may be a compound capable of activating a proteolytically inactive polypeptide of SEQ ID NO: 2 or a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 2. In one aspect, the above cell includes an inducer , which is a proteolytically active polypeptide capable of removing inhibitory amino acid residues from the N-terminus of the above polypeptide. The inductor can, for example, be expressed by recombinant means known to specialists in this field of technology. Alternatively, the inducer can be isolated from a cell, for example, clostridial cells.

The present invention also relates to the use of a nucleic acid molecule of the invention for the production of a proteolytically active polypeptide of the invention.

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

The term "chemical synthesis" refers to the synthesis of polypeptides by chemical means. Such methods are considered, for example, in Nilsson et al., Ann. Rev. Biophys. Biomol. Struct. 2005.34: 91-118. The term "purification of a polypeptide" means the removal from a mixture containing the polypeptide of the invention of compounds other than the above polypeptide. The term also means the removal of the polypeptide of the invention from a mixture comprising compounds other than the above polypeptide. In a particular aspect, the term refers to the separation of a proteolytically active polypeptide from its proteolytically inactive precursor.

Nucleic acid can be translated in a cell or in a cell-free system. Various cell-free translation systems are available to those skilled in the art. The present invention includes, for example, translation in a cell-free protein translation system comprising a rabbit reticulocyte lysate, a wheat germ lysate, an E. coli lysate, or other cellular lysates, for example, lysates derived from C. botulinum and the like. Also included is the translation of the polypeptide of the invention from the nucleotide sequence of the invention or from the vector of the invention. Transcription can be regulated or controlled by one or more heterologous regulatory elements or homologous regulatory elements. Also included in this aspect of the present invention is translation in wild-type cells, i.e. cells isolated from natural sources, such as any known isolates of C. botulinum, C. butyricum, C. baratii and C. tetani . In a particular aspect, the above cells are C. botulinum Hall strain (ATCC 3502). Various standard tools and methods are available to those skilled in the art for delivering nucleic acid or vector molecules to a cell and for expressing the polypeptide of the invention as a recombinant protein in the cell. Moreover, many standard techniques for isolating polypeptides from cells or cell lysates or from cell-free expression systems are known to the person skilled in the art (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 tools and methods can be used in the methods of the present invention.

The first polypeptide of the invention can be translated from a nucleic acid molecule encoding a proteolytically active polypeptide. SEQ ID NO: 26 is an example of such a nucleic acid molecule. Alternatively, the aforementioned nucleic acid molecule may encode a precursor polypeptide that is proteolytically inactive, but which can be converted into a proteolytically active polypeptide of the invention. SEQ ID NO: 27 is an example of such a nucleic acid molecule. A proteolytically inactive precursor is also called an “inactive BoNT hydrolase”, abbreviated iBH. This proteolytically inactive polypeptide can, for example, be activated during or after translation or upon contact, for example, of the aforementioned proteolytically inactive polypeptide with a protease capable of removing inactivating amino acid residues from the N-terminus of the proteolytically inactive polypeptide. An example of a proteolytically inactive polypeptide is a polypeptide represented by the sequence SEQ ID NO: 2. Another example is a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence SEQ ID NO: 2. The term "inactivating amino acid residues at the N-terminus" in one aspect, relates to the first 248 a.k. residues of the above polypeptide. In another aspect, the term refers to a fragment of up to 10 a.k., 50 a.k., 100 a.k., 150 a.k., 200 a.k., 250 a.k. residues of the above polypeptide. Any of these polypeptides are useful in the method of the invention for the production of a proteolytically active polypeptide. In one aspect, a protease capable of removing inactivating amino acid residues from the N-terminus of a given polypeptide is isolated, for example, from Clostridium botulinum, Clostridium butyricum, Clostridium baratii and Clostridium tetani . In another aspect, a protease capable of removing the above inactivating amino acid is obtained by obtaining a fractionated or unfractionated lysate of the above cells. Inactivating amino acid residues can be removed by contacting the proteolytically inactive polypeptide with the above lysate and incubation until the proteolytically inactive polypeptide is converted to the proteolytically active polypeptide.

In another aspect of the method of the invention, the polypeptide is translated in a cell. The cell may be a prokaryotic or eukaryotic cell. In one aspect, the cell is selected from E. coli, B. subtilis, or yeast. Also included in the present invention is the translation of the polypeptide of the invention in a wild-type cell, i.e. in a cell isolated from natural sources, such as any known isolate of Clostridium botulinum, Clostridium butyricum, Clostridium baratii and Clostridium tetani . In a particular aspect, the aforementioned cell is C. botulinum Hall strain (ATCC 3502). In another particular aspect, the aforementioned cell is a cell of the subject invention described above.

Translation products obtained by the method of this invention can be purified by various means known to those skilled in the art (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). Typical methods for purifying a polypeptide of the invention may include centrifuging a cell lysate, precipitating proteins with ammonium sulfate, resuspending proteins, centrifuging resuspended proteins, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography and the like. Several combinations of such steps in different order may be useful for purification of the polypeptide of the invention. A preferred method for purifying a polypeptide of the invention is described in Examples that illustrate the invention.

In one aspect, the purification step comprises binding the polypeptide of the invention to a solid support. The term “solid support” refers to a matrix including, for example, silicone, crosslinked dextran, crosslinked polyacrylamide, or crosslinked agarose and the like. Also included are, in particular, polypeptides, glass, polystyrene, polypropylene, polyethylene, polyethylene glycol (PEG), dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbro and magnetite. The solid support in an aspect of the invention is a polysaccharide matrix selected from the group consisting of Sepharose, Sephadex, agarose, sephacel, microcellulose and alginate beads. In another aspect, the aforementioned solid support may consist of glass beads and / or polypeptide matrices.

In one aspect, the solid support is coupled to an antibody of the invention. The term “coupled” means, in one aspect, stably bound or stably associated. In another aspect, “coupled” includes interactions, such as indirect and direct, irreversible and reversible, physical and chemical, electrostatic and / or covalent bonds. In one aspect, the antibody is covalently coupled, either directly or via a linker molecule, to a solid support. An antibody may be coupled to the aforementioned solid support via a linker, including low molecular weight compounds and peptide (or polypeptide) linker molecules. A solid support may in fact have any possible structural configuration or organization, provided that it provides the ability of the bound antibody to bind to its antigen. Thus, the matrix or solid substrate can be spherical, as in balls, or cylindrical, as on the inner surface of the tube or on the outer surface of the stick. Alternatively, the surface may be irregular or flat, such as a sheet or test strip.

The above antibody bound to a solid support can be used, for example, in the production method of the present invention or in the diagnostic method. In one aspect, the aforementioned production method may include an affinity chromatography step in which the above affinity chromatography is based on an antibody bound to a solid support. In one embodiment of the invention, the aforementioned antibody is an antibody that specifically binds to a proteolytically active polypeptide of the invention. In another embodiment of the invention, the aforementioned antibody is an antibody that specifically binds to a proteolytically inactive polypeptide of the invention.

In another aspect, a method of manufacturing a proteolytically active polypeptide of the invention comprises purifying the polypeptide of the invention from a mixture containing additional components. Cleaning can be based, for example, on polarity, electric charge and size. Thus, the method may, in one aspect, include one or more separation steps selected from the group consisting of normal phase HPLC, reverse phase HPLC, hydrophilic chromatography (HILIC), hydrophobic chromatography (HIC), ion exchange chromatography (IEC), including anion exchange chromatography and cation exchange chromatography, gel filtration chromatography (SEC), gel permeation chromatography (GPC).

In another aspect, the above purification comprises the steps of (a) separation by anion exchange chromatography, (b) separation by gel filtration chromatography, (c) separation by hydrophobic chromatography and (d) separation by gel filtration chromatography.

One or more fractions taken from a chromatographic column can be concentrated, for example, by precipitation or ultrafiltration.

In one aspect, the present invention relates to a composition comprising a proteolytically active polypeptide of the invention. Using the method disclosed in the present description, it is possible to obtain a proteolytically active polypeptide, which is the subject of the present invention, in which there is essentially no proteolytically inactive polypeptide. In other words, the method of the invention provides a proteolytically active polypeptide and a composition that does not include a significant amount of impurities of the inactive protein precursor protein of the polypeptide of the invention. It is assumed that the composition does not contain a significant amount of impurities or does not contain a significant amount of a proteolytically inactive polypeptide precursor if less than 5% of the proteolytically inactive precursor can be determined using the detection method based on Western blot, while the above 5% relate to proteolytically inactive precursor relative to the total amount of proteolytically active and inactive polypeptide. In another aspect, the above composition is substantially purified and comprises at least 50% of the proteolytically active polypeptide of the invention, while the above 50% relate to the amount of proteolytically active precursor relative to the total amount of protein contained in the composition. In another aspect, the above composition is substantially purified and comprises at least 75%, 80%, 90%, or at least 98% of a proteolytically active polypeptide.

In another aspect, the present invention also relates to a polypeptide, which can be obtained by a method of manufacturing a proteolytically active polypeptide, as described above in the present description and illustrated in the Examples. The above proteolytically active polypeptide in one aspect is a proteolytically active polypeptide with a polypeptide sequence of SEQ ID NO: 1. In another aspect, the proteolytically active polypeptide is a polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 1. In another aspect, proteolytically an active polypeptide is a polypeptide that comprises a polypeptide sequence having at least 50% sequence identity with the sequence w SEQ ID NO: 1. The term "polypeptide which is obtained" in this context refers in one aspect to a polypeptide which is translated from the nucleic acid, which is the subject of the present invention. The polypeptide may be subsequently subjected to post-translational modifications, such as acetylation, alkylation, amidation, addition of amino acids, removal of amino acids, glycosylation, oxidation, S-glutathionylation, phosphorylation, sulfation, proteolytic processing and the like. Moreover, the polypeptide can bind a metal ion, 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⁺ .

The present invention also relates, in one aspect, to an antibody that specifically binds to a polypeptide of the invention. The term “antibody” as used herein includes a monoclonal antibody, a polyclonal antibody, a single chain antibody, a human, humanized, primatized or chimerized antibody, a bispecific antibody, a synthetic antibody, chemical or enzymatically modified derivatives, a fragment of any of the above antibodies or aptamers consisting of those found in vivo and / or chemically modified nucleic acids. Fragments of the above antibodies include fragments F (ab ’) 2, F (ab), Fv or scFv, or chemically or enzymatically modified derivatives of any of these fragments.

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

In another aspect, the epitope is formed by amino acid residues located at the N-terminus of amino acid 249 of a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 2. In another aspect, the above epitope is deleted from a proteolytically inactive polypeptide described in the present description, through proteolytic processing.

In another aspect, an epitope to which a specific antibody of the invention is an epitope located at the N-terminus of a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence SEQ ID NO: 1. The term “N-terminus” "In this aspect of the invention relates to the field of a polypeptide comprising 50 N-terminal amino acid residues of the above polypeptide sequence, preferably 25 N-terminal amino acid residues of the above olipeptidnoy sequence. In a particular aspect, the term refers to 14 N-terminal amino acid residues. The term "epitope" in this context refers to an antigenic determinant that is recognized by the antibody of the present invention. In one aspect, the epitope is a linear epitope, in another aspect, the epitope is a conformational epitope. In a particular aspect, the antigenic determinant consists of a peptide having the amino acid sequence of the N-terminus of a proteolytically active polypeptide of the invention, in which the aforementioned peptide can be from 7 to 14, preferably 8, 9, 10, 11, 12, 13 or 14 amino acid residues.

The term “specifically binds” or “specific binds to” in one aspect means that the antibody of the present invention does not cross-react substantially with other epitopes on the polypeptide of the present invention or on other polypeptides in general. Epitope specificity is an important characteristic of an antibody of the invention. The specificity of the antibody with respect to the proteolytically active polypeptide compared to the proteolytically inactive will be in one aspect at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. Specific binding can be investigated by various well-known techniques, including, for example, competitive studies. Another important characteristic is the sensitivity of the antibody. The sensitivity in one aspect of the present invention will be at least 70%, at least 80%, at least 90%, at least 95% of the epitope included in the associated sample. Sensitivity can be investigated by well-known techniques. Specialists in the art will be able to determine the working and optimal research conditions for each determination through routine experiments. Conventional binding assay techniques include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, isothermal microcalorimetry, BIACORE® (surface plasmon resonance, SPR) studies or other surface adsorption methods. The BIACORE® SPR system measures antigen-antibody interaction. The SPR response reflects a change in the concentration of masses on the surface of the detector upon binding or dissociation of the analytes. Based on SPR, BIACORE® measurements in real time directly track interactions as they occur, see BIAapplications Handbook, version AB (1998 edition), BIACORE® code No: BR-1001-86; BIAtechnology Handbook, version AB (reprint 1998), BIACORE® code No: BR-1001-84. Binding properties, such as the sensitivity of an antibody of the present invention, can mainly be determined in binding studies using immobilized antigen (ligand) present on the surface of the sensor. The antibody to be analyzed (analyte) will be presented in the mobile phase, i.e. in solution. In some cases, the antigen is indirectly attached to the surface through binding to another immobilized molecule called a capture molecule. When an antibody is injected in discontinuous portions through a surface with immobilized antigens, three phases can generally be distinguished: (i) the association of the antibody with the antigen during injection of the sample; (ii) an equilibrium or stable state during injection of the sample in which the level of antibody binding is balanced by dissociation from the antibody-antigen complex; (iii) dissociation of the antibody from the surface when the buffer flows. It should be understood that such a study can alternatively be carried out with immobilized antibodies to be investigated, and an antigen containing the solution as a mobile phase. The association and dissociation phases provide information on the kinetics of the interactions of the analyte with the ligand (k _a and k _d , complex formation and dissociation rates, k _d / k _a = K _D ). The equilibrium phase provides information on the affinity of the interactions of the analyte with the ligand (K _D ). In an aspect of the invention, the antibody of the invention has a K _{D of} less than 0.5 μM, in another aspect less than 0.05 μM, in another aspect less than 0.02 μM.

An antibody in the context of the present invention can be produced using methods described, for example, in Harlow and Lane, 1988 (Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988). Monoclonal antibodies can be made using techniques originally described in Kohler & Milstein, 1975 (Kohler & Milstein 1975, Nature 256: 495) and Galfre & Milstein, 1981 (Galfre & Milstein 1981, Meth Enzymol 73: 3). The above techniques include fusion of murine myeloma cells with spleen cells obtained from immunized mammals. Antibodies can subsequently be improved using techniques well known to those skilled in the art. For example, surface plasmon resonance as used in the BIACORE® system can be used to increase the efficiency of phage antibodies that bind to the above epitope within the polypeptide of the invention (cf. Schier et al., 1996, Human Antibodies Hybridomas 7: 97; Malmborg et al., 1995, J. Immunol Methods 183: 7).

In one aspect of the invention, an antibody is produced using a peptide comprising or consisting of the above epitope. The peptide can be produced, for example, by synthetic means or by recombinant expression. Alternatively, an antibody of the invention may be produced using a naturally occurring proteolytically active or inactive polypeptide of the invention. In the latter case, it should be understood that the resulting antibodies must be further investigated for specificity for the polypeptide of the present invention. In another aspect of the invention, the monoclonal antibody of the invention is produced using the polypeptide of the invention that can be treated with a detergent to make the epitope immunologically available. However, it should be understood that in the case when the antibody should be directed to a conformational epitope, such a detergent treatment should not be carried out. In a further aspect, immunostimulating agents, such as cochlear lymphocytic hemocyanin (KLH), can also be used in such a process, especially when using a synthetic peptide.

The antibody of the present invention can be used, for example, for affinity chromatography, immunoprecipitation and immunolocalization of a polypeptide of the invention, as well as for monitoring the presence of the aforementioned polypeptide in samples or in recombinant organisms. Moreover, the antibody of the present invention can be used in a detection method or in a diagnostic method. In a particular aspect, the antibody of the invention is used in a Western blot or in an enzyme-linked immunosorbent assay (ELISA). Also, the antibody of the present invention can be used for therapeutic purposes. In particular, the antibody can be used to inhibit the activity of a proteolytically active polypeptide of the invention. Thus, the antibody of the invention also has a variety of therapeutic uses as described below.

The present invention also relates to the use of a proteolytically active polypeptide of the invention in a method for proteolytically processing a polypeptide. In one aspect, the present invention relates to a method for producing a proteolytically processed polypeptide comprising the step of bringing (a) a first polypeptide, said first polypeptide, which is the subject polypeptide of the present invention, into contact with (b) a second polypeptide, said second polypeptide, which is exposed proteolysis of said first polypeptide, wherein said contact results in proteolytic processing of said second polypeptide to at least two cleavage products.

The present invention also relates to the use of Lys-N and / or Lys-C and / or arginine endopeptidases (Arg-C endoproteases, LeR) from Lysobacter enzymogenes (ATCC 29487) (Wright DS, Graham LD, Jennings PA. Biochim Biophys Acta. 1998 Dec 22; 1443 (3): 369-74). Moreover, the use of plasmin and / or omptin (OmpT) associated with a serine protease membrane that cleaves motifs (Arg / Lys) - (Arg / Lys) (K. Sugimura and T. Nishihara. J. Bacteriol. 170 (also included) 1988), pp. 5625-5632) in a CNT proteolytic processing method such as BoNT / A. In one aspect, the present invention relates to a method for producing a proteolytically processed polypeptide, comprising the step of bringing (a) a first polypeptide, said first polypeptide, which is Lys-C or Lys-N, into contact with (b) a second polypeptide, said second polypeptide, which is exposed proteolysis of said first polypeptide, wherein said contact results in a proteolytic processing of said second polypeptide to at least two cleavage products, and wherein the second polypeptide is single stranded BoNT / A. The term “Lys-C” refers to the 33 kDa Lys-C serine endoprotease from Lysobacter enzymogenes (lysyl endopeptidase, LeK, Genbank number Q7M135), which specifically cleaves peptide bonds located at the C-terminus of lysine, or its homolog having at least least 60% sequence identity. The term “Lys-N” refers to Lys-N metalloendopeptidase isolated from Grifola frondosa and Pleurotis 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 includes homologues of the above protease having at least 60% sequence identity.

This method can be used, for example, for the production of proteolytically processed neurotoxin (CNT) or botulinum neurotoxin (BoNT). The term “BoNT” as used throughout this invention refers to a botulinum neurotoxin and refers to a neurotoxin that can be obtained from C. botulinum , such as serotype A, B, C1, D, E, F or G BoNTs. Also, the terms “CNT” and “BoNTs” include a recombinant and modified neurotoxin comprising one or more modifications, including chemical modifications or genetic modifications. The term "genetic modification" means a deletion, substitution or addition of one or more amino acid residues. Using the method that is the subject of the present invention, it now becomes possible to obtain compositions of neurotoxins with significantly fewer impurities of an unprocessed or partially processed neurotoxin, since these impurities are effectively processed to a double-stranded neurotoxin. In one aspect, the double-stranded neurotoxin is a native double-stranded neurotoxin in which 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 clostridia.

The term "contact" in this context refers to bringing at least two different compounds into physical proximity to provide physical and / or chemical interaction of the above compounds. According to the method of the present invention, two different said compounds are, in one aspect, the first and second polypeptide that are included in the solution. Contact is carried out under conditions and for a time sufficient to ensure the interaction of the first and second polypeptides. The term "proteolytically processed polypeptide" in this context refers, in one aspect, to a polypeptide whose polypeptide chain has been hydrolyzed or cleaved into one or more peptide bonds. In another aspect, the term refers to a polypeptide that has been proteolytically cleaved by an endoprotease or endopeptisade. In another aspect, the term refers to a polypeptide that has been cleaved to a level of at least 50%. In another aspect, the aforementioned proteolytically processed polypeptide is a second polypeptide. In another aspect, at least 60%, 70%, 80%, 90%, or 95% are proteolytically processed.

The term "first polypeptide" as used herein refers to a polypeptide of the invention, i.e. proteolytically active or activated polypeptide, also called "active BoNT hydrolase." Since the active BoNT hydrolase can be obtained from the supernatant of C. botulinum, it was originally called the native BoNT hydrolase, abbreviated “nBH”. However, the terms “first polypeptide” and “nBH” also refer to BoNT hydrolases that can be obtained from other sources. The term "second polypeptide" in this context refers to a substrate of the above first polypeptide. The term “susceptible to proteolysis” refers to a property or requirement of a second polypeptide and is used herein to mean that the second polypeptide can be proteolytically cleaved by the aforementioned first polypeptide. In other words, the term “susceptible to proteolysis” means that the second polypeptide includes a protease recognition and cleavage site, allowing it to act as a substrate for the first polypeptide. A “second polypeptide” is a substrate of the first polypeptide and is proteolytically processed to two or more cleavage products. Using the assay described above, one skilled in the art can verify whether a given polypeptide is a substrate of a first polypeptide and, thus, a “second polypeptide” as defined by the present invention. The term “at least two cleavage products” includes, for example, up to two, three, four, five, and up to six cleavage products.

This method can be used, for example, to obtain a pharmaceutical composition comprising clostridial neurotoxin, or for the formation of polypeptide fragments used in the mass spectrometry method. The first polypeptide and the second polypeptide can be contacted at different stages during the production of the proteolytically processed polypeptide. In one aspect, the step of contacting the first polypeptide and the second polypeptide occurs within the cell. In a particular aspect of this embodiment of the present invention, the first and second polypeptides are expressed in the aforementioned cell.

In another aspect, the above contacting step occurs in a cell lysate or in a purified cell lysate. This aspect includes the addition of the first polypeptide to a lysate or to a purified lysate. The first polypeptide can be added at various stages during the purification of the second polypeptide from the cell lysate. For example, the first polypeptide can be added before or after precipitation of the protein, ion exchange chromatography, hydrophobic chromatography and / or gel filtration chromatography. Moreover, the addition of the first polypeptide to the pharmaceutical composition is also included. In this aspect, the polypeptide of the invention is used, for example, to proteolytically cleave a second polypeptide, for example, to activate a second polypeptide that is a therapeutic agent contained in a pharmaceutical composition. It is also contemplated to introduce a first polypeptide to an object for proteolytic processing of the second polypeptide at the object. Administration also includes co-administration of the first and second polypeptides. The method also includes an incubation step under conditions and for a time sufficient to cleave the second polypeptide. In one aspect, the conditions may include adding a buffer selected from the group consisting of 100 mM Tris-HCl, pH 8.0 or phosphate-saline (50 mM Na ₂ HPO ₄ , 150 mM NaCl, pH 7.4). Preferred buffering conditions include 100 mM Tris-HCl, pH 8.0. “Enough cleavage time” can be determined using the assay described above. In one aspect, the aforementioned “sufficient time for cleavage” depends on the level of cleavage that the proteolytically processed polypeptide or its composition should have. In one aspect, the method comprises the step of incubating the first and second polypeptide for at least 30 minutes, 60 minutes, 120 minutes, or at least 240 minutes. In another aspect, the first and second polypeptides 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 comprises the step of incubating the first and second polypeptide at 4 ° C or at 37 ° C. In another aspect, the method comprises an incubation step of up to 1 hour, up to 2 hours, 4 hours, 6 hours, 10 hours or up to 16 hours.

In one aspect, the polypeptide chain of the aforementioned second polypeptide comprises a sequence selected from any of SEQ ID NO: 4 to 25. In a more particular aspect, the polypeptide chain of the aforementioned second polypeptide comprises a sequence selected from any of SEQ ID NO: 4 to 25, and in which the second polypeptide is cleaved to the C-terminus of the remainder of the basic amino acid within the above sequence from any of SEQ ID NOs: 4 to 25. The above sequences are the amino acid sequences of a known substrate a proteolytically active polypeptide of the invention. Also in the present description it is shown that the above substrates are cleaved to the C-terminus of the remainder of the basic amino acid contained in the sequence, compare Table 1, columns LC and H _N. In a preferred aspect, the aforementioned second polypeptide comprises a sequence selected from SEQ ID NO: 4 to 10. In another preferred aspect, the aforementioned second polypeptide is BoNT / A or a derivative thereof, including, for example, SEQ ID NO: 3 polypeptide and its derivatives. The term "derivative" as used in accordance with this and other aspects of the invention includes amino acid mutations, such as the addition, substitution, deletion or shortening of one or more amino acid residues.

In one aspect, the second polypeptide comprises a derivative of any of SEQ ID NO: 4 to 25 or SEQ ID NO: 3, wherein the above derivative contains one or more point mutations and / or one or more additional amino acid residues. In another aspect, the aforementioned derivative contains 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. Using an activity assay to determine protease activity as described herein, one of skill in the art can determine whether the aforementioned derivative is processed by a proteolytically active polypeptide of the invention. In another aspect, the derivative comprises a point mutation that changes the remainder of the basic amino acid to the remainder of the non-essential amino acid. In another aspect, the derivative has at least 50% sequence identity with any of SEQ ID NOs: 4 to 25. in another aspect, the derivative or polypeptide comprising the derivative is a substrate of the first polypeptide and is proteolytically cleaved by the first polypeptide. A typical example is a derivative of SEQ ID NO: 3, including, for example, one or more point mutations in the light or heavy chain.

In another aspect, the aforementioned polypeptide comprises (a) a polypeptide sequence having at least 30% sequence identity with the sequence SEQ ID NO: 3 [(BoNT / A ATCC 3502, Genbank number AAA23262)]; or (b) a polypeptide sequence selected from the group consisting of tetanus endotoxin, protein coagulation cascade factor X or prothrombin (factor II), digestive pancreatic 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 particular aspect, the sequence identity of the above sequence of the second polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 3 is determined based on amino acid positions 420 to 466 of SEQ ID NO: 3, in another aspect, the above sequence identity is determined based on any from SEQ ID NO: 4 to 25. In other words, the above aspect relates to a second polypeptide comprising a polypeptide sequence that has, for example, 30% sequence identity sequences with a polypeptide sequence between amino acid positions 420 to 466 of SEQ ID NO: 3, or at least 30% sequence identity with the polypeptide sequence of any of SEQ ID NO: 4 to 25. The polypeptide, as defined, can , for example, obtained from C. botulinum, C. tetani or C. sporogenes . The above second polypeptide may 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, replacement, deletion or shortening one or more amino acid residues. Included are, for example, derivatives lacking, for example, the H _C domain of a native neurotoxin or parts thereof, or derivatives with other amino acid residues that replace the H _C domain, as well as derivatives with an additional light chain or other protein load molecule attached to the N-terminus light chain bont.

In another aspect, the second polypeptide may contain additional amino acid residues at the N- or C-terminus or in the internal position. Additional amino acid residues may be flanked by one or more protease cleavage sites. In another aspect, the additional amino acid sequence functions as a detectable marker and / or provides binding to a solid support. An example is a histidine marker or GST (glutathione-S-transferase, glutathione-S-transferase) marker. Another example is the amino acid sequence VPPTPGSAWSHPQFEK containing a Streptag (streptavidin tag, streptavidin marker), preferably attached to the C-terminus.

In another particular aspect, the aforementioned polypeptide is a polypeptide comprising a polypeptide sequence, such as that shown in GenBank with the numbers CBZ04958.1, YP_002805603.1, ZP_02994746.1, YP_001788403.1, YP_001782718.1, ZP_02616437.1, ZP_02661141261146. 1, YP_001255575.1, or a homologue thereof having at least 50% sequence identity.

In another aspect, the biological activity of the aforementioned second polypeptide is modulated by proteolytic cleavage. It is well known to those skilled in the art that the function of many polypeptides can be modulated by proteolytic processing. "Modulate" in this context means to increase or decrease, activate or inactivate. For example, the biological activity of many bacterial neurotoxins increases or is stimulated by the proteolytic processing of a single-stranded neurotoxin to a double-stranded neurotoxin, in which a double-stranded neurotoxin consists of light and heavy polypeptide chains that are covalently linked through a disulfide bridge. The biological activity of a neurotoxin encompasses at least three separate activities: the first activity is a “proteolytic activity” inherent in the light chain of a neurotoxin and is responsible for the hydrolysis of the peptide bond of one or more polypeptides involved in the regulation of fusion of cell membranes. The second activity is the “translocation activity” inherent in the N-terminus of the heavy chain of the processed neurotoxin and is involved in the transport of the light chain through the lysosome membrane into the cytoplasm. The third activity is the “receptor binding activity” inherent to the C-terminus of the heavy chain of the processed neurotoxin and is involved in the binding and capture 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 increased proteolytic activity.

The biological activity of clostridial neurotoxin can be measured using various tests that are known to specialists in this field of technology. These tests determine one or more of the activities mentioned above. For example, a study of a 50% lethal dose (LD ₅₀ ) in mice or an ex vivo study of the phrenic nerve of the right or left dome of the diaphragm in mice (MPN) as described by Pearce et al., 1994 (Pearce LB, Borodic GE, First ER, MacCallum RD (1994), Toxicol Appl Pharmacol 128: 69-77) and Habermann et al., 1980 (Habermann E, Dreyer F, Bigalke H. (1980), Naunyn Schmiedebergs Arch Pharmacol. 311: 33-40) to determine the toxic effect the study drug neurotoxin on a living organism or an isolated neuromuscular preparation. To establish a toxic effect in the LD ₅₀ study, the neurotoxin must be biologically active and have each of the above activities mentioned above. Moreover, various other studies are available, for example, to determine whether a neurotoxin or a light chain of a neurotoxin is proteolytically active. Such studies, for example, are based on the contact of BoNT / A with SNAP-25. Alternatively, a peptide representing the SNAP-25 cleavage site in which the peptide can be labeled to facilitate detection can be used. In a preferred aspect, the biological activity is determined using the MPN assay described below.

In another aspect, the aforementioned first polypeptide is activated by proteolytic processing of an inactive precursor polypeptide, the above 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 proteolytically inactive, while its N-terminated variant They are proteolytic activity. The present invention also provides the use of a proteolytically inactive polypeptide in the methods described herein. The proteolytically inactive polypeptide described herein can be activated, for example, by removing a fragment from the N-terminus or the entire N-terminus including amino acid residues 1 to 248 of SEQ ID NO: 2. In one aspect, the N-terminus is removed by a protease, in in another aspect, the N-terminus is removed by autoproteolysis of SEQ ID NO: 2. 60% sequence identity refers to sequence alignment relative to full length NT02CB1447.

In another aspect, the subject invention of a method for producing a proteolytically processed polypeptide comprises the step of purifying a proteolytically processed second polypeptide or at least one or more cleavage products thereof. Purification of C. botulinum expressing BoNT / A can be performed, for example, as is generally described in the prior art (DasGuppa 1984, Toxicon 22, 415; Sathyamoorthy 1985, J Biol Chemistry 260, 10461). In particular, purification of a neurotoxin may include one or more precipitation and extraction steps, one or more concentration steps, and other specific chromatographic steps. Recombinant single chain BoNT / A and its purification are described in the prior art (Rummel et al., 2004, Mol Microbiol. 51: 631-43).

In a preferred embodiment of the present invention, the clostridium strain is C. botulinum , for example, forming BoNT / A or a derivative thereof. For fermentation, the process described by DasGuppa BR et al. in Toxicon, vol. 22, No.3, p. 414-424, 1984. Thus, 0.5% yeast extract and 0.6% autoclaved yeast mass are added to a 2% medium of NZ-amine type A and the pH is adjusted to 7.2 with 4N NaOH and then obtained in this way Wednesday autoclave. Separately, autoclaved glucose (30% by weight per volume) can be added to this medium to achieve a final concentration of glucose in the medium of 0.5%. Incubation can be carried out, for example, at 37 ° C without stirring, while the fermentation is interrupted, for example, after 96 hours. In the framework of the present invention, in addition to the periodic fermentation described previously, it is also possible to carry out semi-continuous fermentation, repeated periodic fermentation or continuous fermentation.

After the final fermentation and separation of the fermentation medium from the cells with the fermentation medium, the first precipitation can be carried out in order to remove large proteins. Preferably, the precipitation is acid precipitation. Reaction conditions for such acid precipitation are known to those skilled in the art. Typically, 1.5 M H ₂ SO ₄ can be used to acidify the supernatant to a pH of 3.5. Centrifugation is usually carried out for 20 minutes at 2400 × g at 4 ° C. The precipitate obtained by centrifugation can be washed with water, preferably repeatedly. The precipitate can then be extracted with 0.1 M buffer consisting of citric acid and trisubstituted sodium citrate, pH 5.5, for example, for an hour. Then, another centrifugation step can be carried out, for example, at 9800 × g for 20 minutes at 4 ° C. The precipitate obtained, if necessary, can be extracted again, as described previously. The supernatant after extraction and both supernatants, if extraction is repeated, can then be subjected to protamine sulfate precipitation. Precipitation can continue overnight, for example, at 8 ° C. Then the precipitate can be centrifuged, for example, for 20 minutes at 4 ° C and 12000 × g. The supernatant after centrifugation can be subjected to precipitation, such as precipitation with ammonium sulfate, during which other large proteins will be removed. After the precipitation step with ammonium sulfate, a centrifugation step can be added and then the precipitate thus obtained can be re-dissolved and, if necessary, dialysis performed. With the extract, preferably dialyzed and again centrifuged, a series of chromatographic steps can be performed to purify the neurotoxin. Each chromatographic step serves to remove impurities such as protamine sulfate, the remaining DNA, portions of smaller and medium sized proteins, as well as hemagglutinins from the protein complex of botulinum neurotoxin. For this purpose, one or more chromatographic steps may be used in a preferred embodiment of the invention. If necessary, the eluate, for example, from the last chromatographic stage, you can filter and add suitable adjuvants. In one aspect, the filtration is carried out in reaction containers, which can then be subjected to a lyophilization step.

The present invention also relates to a composition that can be obtained by the subject invention of a method for producing a proteolytically processed polypeptide. In one aspect, the above composition comprises a mixture of a processed and unprocessed second polypeptide, in which the above mixture may contain less than 5%, 4%, 3%, 2%, or less than 1% of the unprocessed second polypeptide. In an aspect of the above composition, the aforementioned second polypeptide is BoNT or a derivative thereof. BoNT may, for example, be selected from the group consisting of BoNT serotypes A, B, C, D, E, F and G, including their derivatives. The composition may be, for example, a liquid or solid composition and may contain one or more carriers, adjuvants and / or excipients.

In another aspect, the present invention also relates to a method for the manufacture of a medicament, i.e. a pharmaceutical composition comprising the steps of the aforementioned method and a further step of releasing the purified double-stranded neurotoxin as a medicine. In one aspect, the aforementioned drug comprises a mixture of a processed and unprocessed second polypeptide, in which the above mixture contains less than 5% of an unprocessed second polypeptide. In preferred embodiments of the invention, the mixture comprises less than 4%, 3%, 2%, or less than 1% of an unprocessed second polypeptide.

The present invention also relates to various medical uses of the compounds disclosed in the present description:

In one aspect, the present invention relates to a proteolytically active polypeptide according to the present invention for use as a medicine or in a pharmaceutical composition.

In another aspect, the present invention relates to a composition in accordance with the present invention for use as a medicine or in a pharmaceutical composition.

In another aspect, the present invention relates to an antibody, in accordance with the present invention, for use as a medicine or in a pharmaceutical composition.

In another aspect, the present invention relates to an inhibitor, in accordance with the present invention, for use as a medicine or in a pharmaceutical composition.

In particular, the present invention relates to a pharmaceutical composition comprising a polypeptide of the invention, an antibody of the invention, a composition of the invention, or an inhibitor of the invention.

The term "composition" in this context refers to any composition produced in solid, liquid, aerosol (gaseous) form and the like. The above composition includes, for example, a therapeutically active compound of the invention, optionally together with suitable excipients, such as solvents or carriers or other ingredients. In one aspect, the therapeutically active compound is a proteolytically active polypeptide of the invention. In another aspect, the therapeutic compound is a proteolytically processed second polypeptide as described above, such as a double-stranded neurotoxin. In another aspect, the therapeutically active compound is an antibody of the invention. In another aspect, the therapeutically active compound is an inhibitor of a proteolytically active polypeptide of the invention.

In this context, auxiliary compounds, i.e. compounds that do not affect the effects of the compound of the present invention when the composition is used for the intended purposes, and other ingredients, i.e. compounds that have an additional effect or modulate the effect of the compound of the present invention. Suitable solvents and / or carriers depend on the purpose for which the composition is to be used and on other ingredients. One of skill in the art can immediately determine such suitable solvents and / or carriers.

The carrier (s) must be acceptable in terms of compatibility with other ingredients of the formula and must be harmless to its recipient. Used pharmaceutical carrier may include a solid carrier, gel or liquid. Examples of solid carriers are lactose, calcium sulfate, sucrose, talc, gelatin, agar, pectin, gum, magnesium stearate, stearic acid and the like. Examples of liquid carriers are phosphate-saline solution, syrup, oil, water, emulsions, various types of moisturizing agents, and the like. Similarly, the carrier or solvent may include retention materials well known in the art, such as glycerol monostearate or glycerol distearate alone or with wax. The above suitable carriers include those mentioned above and other carriers well known in the art, see, for example, Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

The solvent (s) are chosen so as not to interfere with the biological activity of the combination. Examples of such solvents are distilled water, saline, Ringer's solutions, dextrose solution and Hanks, in addition, the pharmaceutical composition or formula may also include other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

In one aspect, a pharmaceutical composition in this context includes a biologically active neurotoxin obtained by the method of the present invention, optionally one or more pharmaceutically acceptable carriers. The active neurotoxin may be present in liquid or lyophilized form. In one aspect, the aforementioned compound may be present together with glycerol, protein stabilizers (e.g., human serum albumin (HSA)) or non-protein stabilizers, such as polyvinylpyrrolidone or hyaluronic acid. The pharmaceutical composition in one aspect is administered topically. The commonly used method of drug administration is intramuscular, subcutaneous (near the glands) administration. However, depending on the nature and mechanism of action of the compound, the pharmaceutical composition may also be administered by other methods. The double-stranded neurotoxin polypeptide is an active ingredient in the composition, and in one aspect, it is administered in conventional release forms prepared by combining a drug with standard pharmaceutical carriers in accordance with conventional procedures. These procedures may include mixing, granulating and compressing or dissolving the ingredients in accordance with the target drug. It should be understood that the form and nature of the pharmaceutically acceptable carrier or solvent is dictated by the amount of active ingredient with which it will be combined, the route of administration and other well-known variables.

A therapeutically effective dose refers to the amount of a neurotoxin compound that will be used in a pharmaceutical composition of the present invention that prevents, improves or treats the symptoms accompanying the disease or condition described herein. The therapeutic efficacy and toxicity of a compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, a 50% effective dose (ED 50, 50% effective dose) and a LD ₅₀ (lethal dose for 50% of the population). The dose ratio of therapeutic and toxic effects is the therapeutic index and can be expressed as the ratio of LD ₅₀ / ED ₅₀ .

The medication schedule will be determined by the attending physician and other clinical factors. As is well known in medicine, the dose for any patient depends on many factors, including patient weight, body surface area, age, the compound to be administered, gender, time and route of administration, general health and other drugs that are administered together . Progress can be monitored using periodic evaluations. The pharmaceutical compositions and formulas described herein are administered at least once to treat or improve or prevent the disease or condition listed in this specification. However, these pharmaceutical compositions may be administered more than once.

In a further aspect of the invention, the aforementioned composition is a medicament or a cosmetic composition. In one aspect, the aforementioned medicament comprising a biologically active neurotoxin can be used to prevent and / or treat at least one of the following diseases and disorders: voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign idiopathic blepharospasm, hemifacial spasm and focal spasticity, gastrointestinal disorders, increased sweating, and cosmetic correction of wrinkles, in the following aspect also blepharospasm , oromandibular dystonia, jaw closure dystonia, jaw closure dystonia, bruxism, Meij syndrome, lingual dystonia, eyelid apraxia, open cervical dystonia, anecollis, retrocollis, laterocollis / laryngeal dystonia, dystonia, pharyngeal dystonia, dystonia abductor type, spastic dyspnea, limb dystonia, shoulder dystonia, professional dystonia, cramping spasm, musician spasm, golfer spasm, leg dystonia, hip adduction, knee flexion during abduction of the thigh, knee extension, flexion of the foot, extension of the foot, equinovarus, dystonia of the deformed foot, spontaneous tonic extension of the big toe, flexion of the toe, extension of the toe, axial dystonia, Leaning Tower syndrome, wall segment of the abdomen dystonia, unilateral dystonia, generalized dystonia, X-linked dystonia-parkinsonism, dystonia with cortico-basal degeneration, X-linked dystonia-parkinsonism, late dystonia, dystonia with c of cerebral ataxia, dystonia in Parkinson's disease, dystonia in Huntington’s chorea, dystonia in Gallevorden-Spatz disease, DOPA-induced dyskinesias / DOPA-induced dystonia, late dyskinesias / late dystonia, paroxysmal dyskinesia / dystogenic, kinesia, mild kinesis , myoclonic myokimia, rigidity, weak muscle cramps, hereditary chin tremor, paradoxical activity of masticatory muscles, unilateral spasms of masticatory muscles, hypertension trophic branchial myopathy, masticatory hypertrophy, hypertrophy of the anterior tibial muscle, nystagmus, oscillating supranuclear palsy of the eye, partial continuous epilepsy, planning of surgery for spastic torticollis, paralysis of the vocal cord abductor, non-treatable granulosa dysfunction, miscanthus dysfunction, dysfunction of miscanthus Gilles de la Tourette with stuttering, middle ear myoclonia, protective closure of the pharynx, postlaryngectomy, speech impairment, protective omission upper eyelid, entropion, dysfunction of the sphincter of Oddi, pseudo-achalasia, not associated with achalasia of the esophagus, motor disorders of the esophagus, vaginismus, postoperative immobilization, tremor, dysfunction of the bladder, detrusor-sphincter dysinergia, spasm of the urinary tract, urinary tract sphincter, wrinkles in the corners of the eyes due to the use of cosmetics, asymmetry of the facial muscles, dimples on the chin, muscle stiffness syndrome, tetanus, prostatic hyperplasia, burns treatment of strabismus in cerebral palsy, concomitant mixed paralysis, conditions after surgery for retinal detachment, conditions after cataract surgery, aphakia with myositis strabismus, myopathic strabismus, dissociated vertical displacement, as an aid in strabismus surgery, esotropia, exotropia, achalasia, anal fissures, hyperactivity of exocrine glands, Frey's syndrome, paradoxical lacrimation, increased sweating, axillary, palmar, under venous, rhinorrhea, relative hypersalivation in stroke, in parkinsonism, in spastic conditions in amyotrophic lateral sclerosis, in autoimmune processes in encephalitis and myelitis, multiple sclerosis, transverse myelitis, Devick's disease, viral infections, bacterial infections, parasitic infections hereditary spastic paraparesic postapoplectic syndrome, hemispheric infarction, cerebral stem infarction, spinal cord infarction, migraine, with central nervous system injury , hemisphere lesions, brain stem lesions, spinal cord lesions, central nervous system hemorrhages, cerebral hemorrhages, subarachnoid hemorrhages, subdural hemorrhages, intraspinal hemorrhages, with neoplasia, hemisphere tumors, brain stem tumors, spinal tumors 2000 033863). For details and symptoms see, for example, Jost 2007, Drugs 67 (5), 669 or Dressler 2000 at Botulinum Toxin Therapy, Thieme Verlag, Stuttgart, New York.

In another aspect of the invention, the composition is a cosmetic composition that can be formulated as described for the pharmaceutical composition above. For a cosmetic composition, it is likewise provided that the compound of the present invention is, in one aspect, used in substantially pure form. Cosmetic compositions in the following aspect should be applied intramuscularly. In a further aspect of the invention, cosmetic compositions comprising a neurotoxin can be formulated as an anti-wrinkle solution.

In another aspect, the pharmaceutical composition includes an antibody or inhibitor that is the subject of the present invention. Since the polypeptide of the invention is responsible for the activation of clostridial neurotoxins, the antibody will be useful in reducing the toxic effect observed during infection with clostridia. Thus, the antibody of the present invention can, in one aspect, be used to treat an infection caused by clostridia, including Clostridium perfringens, Clostridium difficile, Clostridium tetani, Clostridium botulinum, Clostridium baratii, Clostridium butyricum, Clostridium sporogenemobloctobloemoblobes, Clostridium sporogenum, Clostridium habromicobloemobes, Clostridium novyi and Clostridium oedematiens . Moreover, the antibody of the present invention, in another aspect, can be used to treat the symptoms associated with the above infection. Moreover, the above antibody can be used in the treatment of a condition or symptom associated with a condition in which the condition is selected from botulism, tetanus, pseudomembrane colitis, gangrene, food poisoning and the like.

In another aspect, the pharmaceutical composition comprises a proteolytically active polypeptide of the invention. The above pharmaceutical composition can be used in one aspect for the proteolytic cleavage of polypeptides involved in coagglutination, in particular for the treatment of patients with hypocoagglutination. In another aspect, the pharmaceutical composition can be used as a fibrinolytic, in particular for the treatment of patients with myocardial infarction, pulmonary embolism, deep vein thromboembolism, i.e. to remove blood clots. The use of a pharmaceutical composition in the treatment of stroke is also contemplated. Moreover, in other aspects, the pharmaceutical composition can be used in the treatment of exocrine pancreatic insufficiency to replace trypsin, chymotrypsin or pepsin. Moreover, in other aspects, the pharmaceutical composition can be used in the treatment of patients with inflammatory reactions, in the treatment of patients with cancer, in particular, for the proteolytic cleavage of tumor antigens exposed to the surface. Moreover, in another aspect, the pharmaceutical composition can be used in the treatment of papilloma.

The present invention also relates to a method for screening an inhibitor, comprising the step of: (a) bringing the proteolytically active polypeptide of the present invention into contact with a known substrate and, optionally, with a putative inhibitor; and (b) determining the effect of the putative inhibitor on the conversion of the substrate to a cleavage product, in which a decrease in the amount of the cleavage product is indicative of the inhibitory effect of the putative inhibitor. In one aspect, the intended inhibitor is a peptide comprising an amino acid sequence selected from any of SEQ ID NOs: 4 to 25, in which at least one basic amino acid from the above amino acid sequence is replaced by a non-basic amino acid. In a further aspect, the aforementioned peptide comprises one or more chemical modifications. In another aspect, the inhibitor is a peptidomimetic of the above peptide. In another aspect, the intended inhibitor is part of a microchip of chemical compounds, i.e. groups of organic chemical compounds. In another aspect, the inhibitor is an antibody of the present invention. This method is applicable for identifying compounds capable of inhibiting a proteolytically active polypeptide of the invention. Initial screening can be based, for example, on a peptide containing an amino acid sequence selected from any of SEQ ID NOs: 4 to 25. Peptides capable of inhibiting the polypeptide of the present invention can be modified to enhance inhibition. Modifications include amino acid substitutions or chemical modifications. Typically, this method is carried out by bringing the polypeptide of the present invention into contact with a known substrate in the presence and absence of a putative inhibitor (method step (a)) and by comparing the effect of the putative inhibitor in converting the substrate to a cleavage product. A decrease in conversion rate in the presence of a putative inhibitor is an indication of an inhibitory effect.

The present invention also relates to an inhibitor of a proteolytically active polypeptide of the invention, in which the aforementioned inhibitor is (a) an inhibitor containing an amino acid sequence, such as that shown in any of SEQ ID NOs: 4 to 25, in which the main the amino acid is replaced by a non-essential amino acid; or (b) an antibody of the invention.

All references cited in this description of the invention are incorporated into this description by reference in its entirety, the disclosure of their content and the disclosure of the content specifically mentioned in this description of the invention.

The figures show:

Figure 1: Study of the activity of fractions collected with HiPrep 16/10 Q FF

5 μl of fractions collected from the HiPrep 16/10 Q FF assay were analyzed for enzymatic activity by incubating 2 μg scBoNTA (lane 2) for 1 h at 37 ° C and subsequent electrophoresis in 10% SDS-PAGE (sodium dodecyl sulfate) . Lane 1: low molecular weight markers (LMW): 116 kDa, 66 kDa, 45 kDa, 35 kDa.

Figure 2: Analysis of the collected fractions of gel filtration chromatography (SEC) (HiLoad 16/60 Superdex 75) for nBH content with a molecular weight of ~ 37.3 kDa by electrophoresis in 12.5% SDS-PAGE.

Fractions 9 to 11 contain the bulk of nBH. (Track 1: LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa).

Figure 3: Electrophoretic analysis in 12.5% SDS-PAGE to determine the purity and concentration of protein from three series of purification of nBH.

Track 1, LMW (116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa); lane 2, nBH batch TE311206 (192 ng / μl mature NT02CB1446 / CBO1444, amino acids 254-594 Genbank number CAL82987.1, molecular weight 38.6 kDa); lane 3, nBH batch TIK301009 (130 ng / μl mature NT02CB1447 / CBO1445, SEQ ID NO: 1, amino acids 249-581 Genbank number CAL82988.1, molecular weight: 37.3 kDa); lane 4, nBH batch TIK280509 (114 ng / μl mature NT02CB1447 / CBO1445, SEQ ID NO: 1, amino acids 249-581 Genbank number CAL82988.1, molecular weight 37.3 kDa).

Figure 4: Report on the analysis of the spectrum obtained using tandem mass spectrometry with electrospray ionization.

The 38.6 kDa nBH protein band of the TE311206 lot was identified as NT02CB1446 / CBO1444 with a Mascot value of 725 and a tandem mass spectrometry peptide sequence coating of 29.6% over the entire open reading frame (ORF). Not a single peptide was identified (gray rectangle, identified MS peptide; red squares, identified amino acid y- / b-ions of the peptide after tandem mass spectrometric fragmentation) obtained from 253 N-terminal amino acids. Analysis of the TE311206 batch by tandem mass spectrometry showed a sequence coverage of 52%, which corresponds to the C-terminal and amino acids 254-594 forming nBH.

Figure 5: Report on the analysis of the spectrum obtained using tandem mass spectrometry with electrospray ionization.

The 37.3 kDa nBH protein band of batch TIK301009 was identified as NT02CB1447 / CBO1445 with a Mascot value of 555 and a tandem mass spectrometry peptide sequence coating of 28.4% over the entire open reading frame (ORF). In addition to one, all identified peptides (gray box, identified MS peptide; red squares, identified amino acid y- / b-ions of the peptide after tandem mass spectrometric fragmentation) were obtained from 333 C-terminal amino acids. TIK301009 batch analysis by tandem mass spectrometry showed a sequence coverage of 49.5%, which corresponds to the C-terminal amino acids 249-581 forming nBH.

Figure 6: Comparison of concentration-dependent proteolytic activity of nBH obtained from three purification series.

A. Electrophoresis in 12.5% SDS-PAGE in an activity study analyzing nBH obtained from the TIK301009, TIK280509 and TE311206 series using the following dilutions of concentrated nBH: 1:10, 1:30, 1: 100, 1: 300, 1: 1000. The study was carried out by incubating 1 μg scBoNT / A and 2 μl distilled water and 1 μl appropriately diluted nBH for 60 min at 37 ° C. For electrophoretic analysis in SDS-PAGE, 3 μl of 4 × Lammley reducing buffer with SDS was added to a final volume of 10 μl. 150 kDa scBoNT / A was digested to 100 kDa of the heavy chain and 50 kDa of the light chain.

B. The optical density of the heavy chain, light chain and scBoNT / A protein bands was quantified, and the sum of the light and heavy chain product bands was divided by the sum of the light chain, heavy chain and scBoNT / A protein bands. A stronger dilution of the first polypeptide reduces the rate of cleavage. The specific proteolytic activity of three different series is almost identical.

Figure 7: Time-dependent cleavage of wild-type scBoNT / A and mutants containing a modified loop by nBH.

A. Modification of the loop sequence. All lysine residues were removed in scBoNTAS Throm and the LVPRGS thrombin recognition sequence was inserted, while in scBoNT Res the loop was devoid of any basic amino acid residues. Shortening the loop to eight small residues or five amino acids with bulky side chains leads to the formation of scBoNTAS (GGSG) ₂ and scBoNTAS FQWYI, respectively. The entire loop was removed in scBoNTAS CGS-C, and the cysteines forming the disulfide bridge were replaced by glycine and serine.

B. Electrophoretic analysis in SDS-PAGE of time-dependent cleavage of wild-type scBoNT / A and mutants.

Wild-type C. scBoNTAS is activated by nBH in a time-dependent manner to the light chain and heavy chain in 120 minutes. The absence of lysines and the insertion of a single arginine residue lengthens the loop cleavage time (scBoNTAS Throm). A loop devoid of any basic residue can still be split (scBoNTAS Res). Shortening the loop to an 8-dimensional peptide, introducing five amino acids with bulky side chains, or removing the entire loop results in the formation of a non-cleavable scBoNT / A.

Figure 8: Tandem mass spectrometric analysis of 50 kDa and 100 kDa cleavage products by cleavage of scBoNT / A using nBH.

A. Analysis of a 50 kDa cleavage product that has been identified as a scBoNT / A light chain with a Mascot of 1460. The C-terminal ascribed peptide covers amino acids G433 to K438, which corresponds to the physiologically observed C-terminus of scBoNT / A light chain.

B. Analysis of a 100 kDa cleavage product that has been identified as a scBoNT / A heavy chain with a Mascot score of 96. The N-terminal assigned peptide covers amino acids A449 to K456, which corresponds to the physiologically observed N-terminus of the scBoNT / A heavy chain.

Figure 9: A. The protein content (mg / ml) of anti-nBH-IgY from three consecutive pools was analyzed by electrophoresis in 12.5% SDS-PAGE. B. ELISA: Nunc Maxisorp F96 microtiter plates were coated with nBH from various lots (500 ng / ml) in phosphate-saline overnight at 4 ° C and then blocked for 1 h with blocking buffer from phosphate-saline containing 0 , 1% Tween-20 and 2% skim skim milk. After washing, dilution of IgY from each pool (10 μg / ml in blocking buffer) was added over 1 h and detected using biotin-labeled donkey immunoglobulins Y against chicken immunoglobulins fused with horseradish peroxidase streptavidin (both from Dianova, Hamburg, Germany) and 3 3 ', 5,5'-tetramethylbenzidine (Sigma).

Figure 10: A. Recombinant expression and isolation of inactive BH 1-581 (63 kDa) by affinity chromatography on immobilized Talon metal ions (IMAC). Electrophoretic analysis in 10% SDS-PAGE affinity chromatography fractions on immobilized Talon metal ions (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa; SS34, clarified lysate; TD, unbound fraction; W, wash fraction; E1-E7, fractions 1 to 7 eluted with imidazole). C. There is no endoproteolysis of scBoNT / A to the light chain (50 kDa) and heavy chain (100 kDa) with recombinant iBH (SEQ ID NO: 2, “E”; 63 kDa) at 37 ° C after 1 h (lane 6) (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa).

Figure 11: The use of purified active BoNT hydrolase (nBH) to obtain proteolytically processed polypeptide

A. 200 μg of recombinant purified scBoNT / A is incubated with 350 ng of purified active BoNT hydrolase for 12 min at 37 ° C. To stop the reaction, nBH was removed by size exclusion chromatography (Superdex 200 10/300 GL column, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min ), and the level of cleavage is analyzed by electrophoresis in 10% SDS-PAGE. B. Fraction 1 (1800 μl) containing ~ 40% processed BoNT / A was incubated with 350 ng of purified active BoNT hydrolase for 15 min at 37 ° C and concentrated to 300 μl by ultrafiltration. To finally stop the reaction, nBH was removed by gel filtration chromatography (Superdex 200 10/300 GL column, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min), and the level of cleavage is analyzed by electrophoresis in 10% SDS-PAGE. C. Fractions 1 and 2 (1800 μl) containing ~ 80% processed BoNT / A are combined and incubated with 120 ng of purified active BoNT hydrolase for 25 min at 37 ° C and concentrated to 300 μl by ultrafiltration. To finally stop the reaction, nBH was removed by gel filtration chromatography (Superdex 200 10/300 GL column, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min), and the level of cleavage is analyzed by electrophoresis in 10% SDS-PAGE. Receive> 95% processed BoNT / A (SEQ ID NO. 3).

The list of sequences shows:

SEQ ID NO: 1: Proteolytically active polypeptide obtained from Clostridium botulinum strain ATCC 3502, GenBank number CAL82988.1, lacking 248 N-terminal amino acid residues

SEQ ID NO: 2: Proteolytically inactive polypeptide obtained from Clostridium botulinum strain ATCC 3502, GenBank number CAL82988.1

SEQ ID NO: 3: BoNT / A ATCC 3502, GenBank Number AAA23262

SEQ ID NO: 4: Loop BoNT / A1

SEQ ID NO: 5: Loop BoNT / A2 / A6

SEQ ID NO: 6: BoNT / A3 Loop

SEQ ID NO: 7: BoNT / A3 Loop

SEQ ID NO: 8: BoNT / A4 Loop

SEQ ID NO: 9: Loop BoNT / A5

SEQ ID NO: 10: Loop BoNT / A7

SEQ ID NO: 11: Loop BoNT / B1 / B4bv / B6

SEQ ID NO: 12: Loop BoNT / B2 / B3

SEQ ID NO: 13: BoNT / B5np Loop

SEQ ID NO: 14: BoNT / C / CD Loop

SEQ ID NO: 15: Loop BoNT / D

SEQ ID NO: 16: BoNT / DC Loop

SEQ ID NO: 17: Loop BoNT / E1-E5

SEQ ID NO: 18: BoNT / E6 Loop

SEQ ID NO: 19: Loop BoNT / F1 / F6

SEQ ID NO: 20: BoNT / F2 / F3 Loop

SEQ ID NO: 21: Loop BoNT / F4

SEQ ID NO: 22: Loop BoNT / F5

SEQ ID NO: 23: Loop BoNT / F7

SEQ ID NO: 24: Loop BoNT / G

SEQ ID NO: 25: TeNT Loop

SEQ ID NO: 26: nucleic acid sequence encoding SEQ ID NO: 1

SEQ ID NO: 27: nucleic acid sequence encoding SEQ ID NO: 2

The following examples illustrate the invention and should not be construed as limiting its scope.

EXAMPLES

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

(1) Reading system / activity assay : For the specific detection and purification of enzymatic activity that hydrolyzes botulinum neurotoxin A (BoNT / A) up to 50 kDa light chain (LC) and 100 kDa heavy chain (HC) in C. botulinum culture supernatants and between by chromatographic steps, we expressed 150 kDa BoNT / A as a single chain (sc) polypeptide in E. coli . Incubation of recombinant scBoNT / A with suitable enzymatic activity (nBH) should result in the formation of 50 kDa LC and 100 kDa HC visualized by reduction electrophoresis in 10-13% SDS-PAGE.

(2) Expression of clostridial protease: One colony of C. botulinum strain ATCC 3502 was inoculated in 100 ml of cardiac extract (BHI) medium and the culture was incubated overnight at 37 ° C. under anaerobic conditions. 10 ml of overnight culture was inoculated in 1 L of BHI medium and incubated under anaerobic conditions for 48-72 hours.

(3) Ammonium sulfate precipitation: 1 L of culture supernatant was collected by centrifugation (4 ° C, 6500 × g, 25 min). Ammonium sulfate was added to a final concentration of 85% (here 575 g), the suspension was stirred for 6 hours at 4 ° C and subsequently centrifuged (4 ° C, 6500 × g, 30 min). The precipitated ammonium sulfate precipitate was dissolved in a small volume (5 ml here) of 50 mM NaP pH 7.5 and dialyzed against 50 mM NaP, 150 mM NaCl pH 7.5. At the end, the dialysate was centrifuged (4 ° C, 40,000 × g, 60 min) and the supernatant was used for ion exchange chromatography.

(4) Ion exchange chromatography (IEC, HiPrep 16/10 Q FF column): the supernatant from (3) (Figure 1, lane 3) was applied to a balanced HiPrep 16/10 Q FF anion exchange column and washed with a buffer containing 50 mM NaP pH 7 5, 150 mM NaCl. The experiment was carried out at a flow rate of 1 ml / min. The activity study was carried out by incubating 5 μl of each second fraction with 2 μg scBoNTA for 1 h at 37 ° C and subsequent electrophoretic analysis in SDS-PAGE (Figure 1). Fractions 6-24 were combined and the volume was concentrated to 3.5 ml by ultrafiltration (Amicon-Ultra MWCO 10,000).

(5) Gel filtration chromatography (SEC, HiLoad 16/60 Superdex 200): Subsequently, the concentrated protein solution from step (4) was applied to a HiLoad 16/60 Superdex 200 column, equilibrated with 50 mM NaP pH 7.5, 150 mM NaCl. Separation was carried out at a flow rate of 1 ml / min. Fractions with a retained volume of between 80 ml and 100 ml were analyzed using an activity assay (1), and suitable fractions containing enzymatic activity (nBH) were combined (~ 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).

(6) Hydrophobic chromatography (HIC, HiTrap Phenyl Sepharose): nBH was coupled to phenylsepharose in buffer A (50 mM NaP pH 7.5, 500 mM ammonium sulfate). Bound nBH was eluted by decreasing the amount of ammonium sulfate in a linear incremental gradient with buffer B (50 mM NaP pH 7.5) at a flow rate of 1 ml / min. All protein containing fractions were analyzed using activity assay (1), and the appropriate fractions were combined and concentrated by ultrafiltration to 3.5 ml. The solution buffer was adjusted to 50 mM NaP pH 7.5; 150 mM NaCl.

(7) SEC (HiLoad 16/60 Superdex 75): Finally, nBH was purified by SEC using a HiLoad 16/60 Superdex 75 column in 50 mM NaP pH 7.5; 150 mM NaCl and a flow rate of 1 ml / min. Fractions with a retention volume between 70 ml and 80 ml were analyzed by electrophoresis in 12.5% SDS-PAGE (Figure 2), and fractions 8-12 containing nBH, which migrates at a level of ~ 37.3 kDa, were combined (~ 10 ml) and concentrated to 1 ml by ultrafiltration.

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

(9) Two batches of nBH (NT02CB1447, 37.3 kDa, Figure 3, lane 3: TIK301009, lane 4: TIK280509) were reproducibly isolated in accordance with the procedure described above. The following modifications to the isolation procedure lead to the formation of nBH isoform NT02CB1446 (38.6 kDa, Figure 3, lane 2, batch TE311206): (i) growing a culture of C. botulinum : 18 hours instead of 48 hours - 72 hours; (ii) a variation of the chromatographic steps: IEC → SEC Superdex 75 → HIC phenylsepharose instead of IEC → SEC Superdex 200 → HIC phenylsepharose → SEC Superdex 75.

Example 2: Identification of the sequence of nBH from C. botulinum  by mass spectrometry (MS)

(1) Tryptic cleavage: Protein bands of approximately 38 kDa (nBH) in SDS-PAGE electrophoresis were excised for tryptic cleavage and discolored by gentle stirring in 50 mM Na ₄ HCO ₃ , 50% acetonitrile for 30 min at 37 ° C. Discoloration was repeated until the spots in the gel became transparent. Acetonitrile (100%) was added and removed after 3 minutes. The spots were then dried in a high-speed vacuum system (Eppendorf, Germany). Trypsin (10 ng / μl) in 50 mM NH ₄ HCO _{3 was} added and incubated on ice for 1 h. Then, the remaining trypsin solution was removed, a small volume of 50 mM NH ₄ HCO ₃ was added and cleavage was performed at 37 ° C overnight. The supernatant was collected and the gel pieces were extracted twice using 5% trifluoroacetic acid, 10% acetonitrile. All liquids were combined, dried under high speed vacuum, and the extracted peptides were stored at 4 ° C.

(2) Tandem time-of-flight mass spectrometry with laser ionization and desorption from a liquid matrix (MALDI-TOF / TOF MS): samples were analyzed in a mass spectrometer for time-of-flight MALDI mass spectrometry (Ultraflex 1 Bruker Daltonik GmbH) in a linear mode with accelerating voltage 25 kV. Masses were detected from 700 m / z to 4500 m / z. Samples (2 μl) were crystallized with 2 μl of a solution of synapinic acid containing 50% acetonitrile and 0.2% trifluoroacetic acid (TFA) directly on a steel substrate for MALDI. 500 laser pulses were collected for each sample.

(3) Peptide Separation by Reverse Phase Chromatography: Peptides were separated by reverse phase chromatography using a nano-HPLC system (Agilent Technologies, Waldbronn, Germany), which consists of an automatic dispenser and a gradient pump. The sample was dissolved in buffer A (5% acetonitrile, 0.1% formic acid) and an aliquot of up to 10 μl was injected into a C18 column (Zorbax SB-C18, 5 μl, 300 A, 0.5 mm inner diameter, length 15 cm) at flow rates of 5 μl / min. After application, the column was washed with buffer A for 15 minutes and the peptides were eluted using a gradient of eluent A and eluent B (70% (v / v) acetonitrile in 0.1% (v / v) formic acid) from 0% to 100% eluent B in 75 minutes

(4) Electrospray ionization interface (ESI) and ion trap mass spectrometry: the HPLC output was directly connected to the source of an nano ESI ion trap mass spectrometer and an Agilent coaxial flow-through liquid atomizer was used (Agilent Technologies). The output capillary was held by an adjacent steel needle and protruded 0.1-0.2 mm from it. Spraying was stabilized with N ₂ as a nebulizer gas (5 L / min). The ionization voltage was set at 4500 V and dry gas was used at 5 psi (34.47 kPa) and 250 ° C. Spectra were recorded using an Esquire3000 + ion trap mass spectrometer (Bruker Daltonik) at a scan speed of 13000 m / z per second. Using ESI in the mode of registration of positive ions, we obtained mass spectra from 50 to 1600 m / z in the scanning mode and data-dependent switching between MS and tandem MS analysis. To improve the quality of the tandem MS spectra, only two precursor ions were selected from the same spectrum for tandem MS analysis and active exclusion was set for 2 min to exclude precursor ions that were already measured.

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

(5) Results:

table 2
nBH identified by MS Track NBH party Protein concentration ORF name Genbank Number A.K. in ORF Molecular Weight [kDa] Mascot Score 2 TE311206 192 ng / μl NT02CB1446
CBO1444 CAL82987.1 254-594 38.6 725 3 TIK301009 130 ng / μl NT02CB1447
CBO1445 CAL82988.1 249-581 37.3 555 4 TIK280509 114 ng / μl NT02CB1447
CBO1445 CAL82988.1 249-581 37.3 609

The 38.6 kDa protein band from lane 2 (nBH batch TE311206) was identified as NT02CB1446 / CBO1444 with a Mascot of 725 and a tandem MS peptide sequence coating of 29.6% throughout the open reading frame (ORF). Not a single peptide derived from 253 N-terminal amino acids was identified (Figure 4). Analysis of the tandem MS batch of TE311206 showed a sequence coverage of 52%, which corresponds to the C-terminal amino acids 254-594 forming nBH.

37.3 kDa protein bands from lane 3 (nBH batch TIK301009) and lanes 4 (nBH batch TIK280509) were identified as NT02CB1447 / CBO1445 with a Mascot of 555 and 609, respectively. In addition to one, all identified peptides are derived from 333 C-terminal amino acids (Figure 5). Analysis of the tandem MS batch TIK301009 showed a sequence coverage of 49.5%, which corresponds to the C-terminal amino acids 249-581 forming nBH.

Example 3: Characterization of the enzymatic specificity of nBH

(1) The concentration-dependent proteolytic activity of nBH obtained from their three series of purification was compared (Figure 6). An activity study analyzing nBH obtained from the TIK301009, TIK280509, and TE311206 series using different dilutions of nBH shows that higher dilutions reduce the rate of cleavage. The proteolytic activity of three different series is almost identical, which indicates that the mature isoform NT02CB1446 (TE311206) exhibits specific activity similar to mature NT02CB1447 (SEQ ID NO: 1).

(2) Time-dependent cleavage of wild-type scBoNT / A and nBH mutants was analyzed using an activity test (Figure 7). wild-type scBoNT / A is activated by nBH in a time-dependent manner to the light chain and heavy chain in 120 minutes by more than 95%. The loop sequence was modified to characterize the cleavage site. In scBoNT / A Throm, all lysine residues were removed and the LVPRGS thrombin recognition sequence was inserted, which lengthens the cleavage path. In scBoNT Res, the loop is devoid of any basic amino acid residues, which greatly delays complete hydrolysis, indicating a strong preference for basic residues like lysine and arginine to recognize nBH at the cleavage site. Moreover, the availability of nBH for the loop is impaired by shortening the loop to eight small residues or five amino acids with bulky side chains (scBoNTAS (GGSG) ₂ and scBoNTAS FQWYI).

(3) A tandem MS analysis of 50 kDa of the digestion product by digesting scBoNT / A nBH showed that the C-terminal ascribed peptide covers amino acids G433 to K438, which corresponds to the physiologically observed C-terminus of the scBoNT / A light chain (Figure 8A). Analysis of the 100 kDa cleavage product, identified as the BoNT / A heavy chain, showed that the N-terminal assigned peptide covers amino acids A449 through K456, which corresponds to the physiologically observed N-terminus of the scBoNT / A heavy chain (Figure 8B). Thus, the isolated nBH leads to the formation of a physiologically processed BoNT / A and preferably hydrolyzes peptide bonds to the C-terminus of lysine and arginine residues.

Example 4: Evolutionary Conservativeness of BoNT Hydrolases and Its Isoforms

Analysis of the protein sequence of SEQ ID NO: 2 (Genbank number CAL82988.1 / YP_001253958.1) revealed three conservative domains. Residues 18-573 correspond to zinc metalloprotease (elastase) or LasB involved in the transport and metabolism of amino acids, with a Blast score of 738. Residues 148-212 correspond to the peptidase propeptide and the YPEB or PepSY domain (Blast score 97). Residues 336-573 are part of the peptidase of the M4 family, including thermolysin, protealysin, aureolysin, and neutral proteases (Blast 803).

Genome sequencing of C. botulinum ATCC 3502 revealed the existence of six ORF coding for nBH isoforms (Sebaihia et al., 2007, Genome Res. 17 (7): 1082-1092). The following genomic data are available for 10 group I strains of C. botulinum , as well as for non-secreting BoNT C. sporogenes , which contain between five and seven ORFs encoding iBH. nBH (SEQ ID NO: 1) has a minimum of 64% amino acid sequence identity with the other 63 isoforms.

Example 5: The formation of antibodies specific for BoNT hydrolase

(1) Formation of IgY: sixteen- week- old chickens [ISA Brown Lohmann Selected Leghorn (LSL), Spreenhagener Vermehrungsbetrieb fur Legehennen GmbH, Bestensee, Germany] were kept in individual cages specially designed for keeping chickens (Ebeco, Castrop-Rauxel, Germany). Food (ssniff Leghuhner-Zucht 1 and 2; ssniff Spezialitaten GmbH, Soest, Germany) and water were available without restriction, and chickens began to lay eggs between the ages of 23 and 25 weeks. Eggs were collected daily, labeled and stored at 4 ° C until further use. All animal care and all experiments were carried out in accordance with local authorities, Berlin (No. H0069 / 03). Chickens were immunized and re-immunized intramuscularly (pectoral muscle, left and right side) only 10 times in 1 year, at intervals of 4 and 8 weeks. The interval used was based on previous work, which showed the absence of explicit memory cells up to at least 3 weeks after immunization (Pei and Collisson, 2005). The applied antigen concentration was approximately 20 μg per injection (nBH). No more than 500 μl of antigen solution was injected per immunization. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent repeated injections. The IgY purification method was adapted from Polson et al. (1980). Briefly, the egg yolk was dissolved 1: 2 with sterile phosphate-saline solution (pH 7.4, Roche, Mannheim, Germany). To remove lipids and lipoprotein, 3.5% (w / v) polyethylene glycol (PEG) 6000 (Roth, Karlsruhe, Germany) was added. After gentle stirring followed by centrifugation (10,000 g for 20 minutes at 4 ° C), the supernatant was removed and solid PEG 6000 was added to a final concentration of 12% (w / v). Then this mixture was centrifuged as described above. The precipitate was dissolved in 10 ml of phosphate-saline solution, PEG was added to 12% (w / v) and the solution was centrifuged. At the end, the precipitate was dissolved in 1.2 ml of phosphate-saline solution, transferred to a microdialysis apparatus (QuixSep, Roth, Germany) and dialyzed against phosphate-saline solution at 4 ° C. Protein content (mg / ml) was analyzed by 12.5% SDS-PAGE electrophoresis (Figure 9A) and measured photometrically at 280 nm and calculated according to the Lambert-Behr law with an extinction coefficient of 1.33 for IgY.

(2) Enzyme-linked immunosorbent assay (ELISA) : Nunc Maxisorp F96 microtiter plates (VWR International GmbH, Darmstadt, Germany) were coated with nBH from different lots (500 ng / ml) in phosphate-saline solution at 4 ° C and then blocked for 1 hr with phosphate-saline blocking buffer containing 0.1% Tween-20 and 2% skim skim milk (Merck, Darmstadt, Germany). After washing, IgY dilution (10 μg / ml in blocking buffer) was added over 1 h and detected using Biotin-labeled donkey IgY against chicken immunoglobulins fused with streptavidin horseradish peroxidase (both from Dianova, Hamburg, Germany) and 3.3 ', 5,5'-tetramethylbenzidine (Sigma). Detectable nBH is shown in Figure 9B.

(3) Western blot : nBH was separated by electrophoresis in 12.5% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Invitrogen GmbH, Karlsruhe, Germany) using standard immunoblot techniques. The membrane was blocked overnight at 4 ° C and incubated with IgY (1: 5000 in blocking buffer) for 1 h. After washing, the membrane was examined with biotin-labeled donkey IgY against chicken immunoglobulins for 30 minutes and developed using alkaline phosphatase and CDP- Star (Perkin Elmer, Waltham, Mass.).

Example 6: Recombinant expression of BoNT hydrolase

(1) Plasmid constructs: portions of a gene encoding native BH (SEQ ID NO: 1) and its propeptide (SEQ ID NO: 2) were amplified by PCR using suitable oligonucleotides and genomic DNA of C. botulinum ATCC 3502 fused to the oligonucleotide encoding His6Tag and inserted into pQE3 (Qiagen), resulting in the expression of plasmids pQ-BH1445H6-249-581 and pQ-BH1445H6-1-581, respectively. Nucleotide sequences were checked by DNA sequencing.

(2) Purification of recombinant proteins: nBH and iBH fused to the carboxy-terminal His6Tag were obtained using E. coli strain M15pREP4 (Qiagen) for ten hours of incubation at room temperature and purified on Talon-Sepharose beads (Clontech Inc.) following the manufacturer's instructions . Fractions containing the desired proteins were combined, frozen in liquid nitrogen and stored at -70 ° C. iBH was isolated as a recombinant protein with a molecular weight of 63 kDa (Figure 10A). The lack of activity of iBH was demonstrated using an activity study: after 1 h at 37 ° C, wild-type scBoNT / A did not hydrolyze to the light chain and heavy chain (Figure 10B).

Example 7 Inhibition of BoNT Hydrolase

(1) Screening of peptide BH inhibitors: peptides based on SEQ ID NOs: 4 to 25 are synthesized without one or more basic residues. Each peptide will be added to the mixture, in accordance with the activity study. A peptide capable of reducing the amount of scBoNT / A processed, lengthening the time required for complete scBoNT / A processing, or blocking scBoNT / A processing, is considered an nBH inhibitor.

(2) Screening of antibody-based inhibitors: antibodies obtained against epitopes derived from nBH, like the IgY from Example 5, are incubated with nBH and subsequently an activity study is performed. An antibody capable of reducing the amount of scBoNT / A processed, lengthening the time required for complete scBoNT / A processing, or block scBoNT / A processing, is considered an nBH inhibitor.

Example 8: Use of Purified Active BoNT Hydrolase (nBH) to Produce a Proteolytically Processed Polypeptide

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

(2) Fraction 1 (1800 μl) containing ~ 40% processed BoNT / A was incubated with 350 ng of purified active BoNT hydrolase for 15 min at 37 ° C and concentrated to 300 μl by ultrafiltration. To finally stop the reaction, nBH was removed by SEC (Superdex 200 10/300 GL column, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min), and quantitative analysis of cleavage is carried out by electrophoresis in 10% SDS-PAGE (Figure 11B).

(2) Fractions 1 and 2 (1800 μl) containing ~ 80% processed BoNT / A are combined and incubated with 120 ng of purified active BoNT hydrolase for 25 min at 37 ° C and concentrated to 300 μl by ultrafiltration. To finally stop the reaction, nBH was removed by SEC (Superdex 200 10/300 GL column, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min), and a quantitative analysis of cleavage is performed by electrophoresis in 10% SDS-PAGE (Figure 11C). Get> 95% processed BoNT / A (SEQ ID NO: 3). An identical fully processed second polypeptide (> 95% processed BoNT / A) is obtained if the second polypeptide is processed in one step for 50 min at 37 ° C (200 μg scBoNT / A was incubated with 350 ng nBH). After incubation for 1 h at 37 ° C, more than 97% of BoNT / A is processed.

--->

LIST OF SEQUENCES

<110> toxogen GmbH

<120> METHODS FOR PRODUCING PROTEOLYTIC PROCESSED POLYPEPTIDES

<130> 757-1

<160> 27

<170> PatentIn version 3.5

<210> 1

<211> 333

<212> PROTEIN

<213> Clostridium Botulinum

<400> 1

Val Gln Gly Gln Ser Val Lys Gly Val Gly Lys Thr Ser Leu Asp Gly

1 5 10 15

Leu Val Asn Ile Asp Val Thr Tyr Gly Asn Gly Lys Tyr Tyr Leu Lys

            20 25 30

Asp Ser Asn Lys Asn Ile Tyr Leu Tyr Asp Leu Lys Asn Gln Val Asp

        35 40 45

Glu Tyr Asp Leu Tyr Asn Tyr Leu Ser Arg Pro Asn Tyr Lys Gln Ile

    50 55 60

Leu Met Ser Lys Ser Glu Leu Ile Ser Asn Tyr Asn Asn Asn Phe Ile

65 70 75 80

Ala Asn Asn Gln Val Asn Ser Val Asp Ala Tyr Val Asn Thr Asn Lys

                85 90 95

Thr Tyr Asp Tyr Tyr Lys Asn Lys Leu Asn Arg Asn Ser Ile Asp Asn

            100 105 110

Lys Gly Met Asn Ile Asn Gly Phe Val His Val Gly Arg Asn Tyr Gly

        115 120 125

Asn Ala Phe Trp Tyr Gly Pro Tyr Asp Gly Met Phe Phe Gly Asp Gly

    130 135 140

Asp Gly Ile Tyr Phe Ser Ser Leu Ala Lys Ser Leu Asp Val Val Gly

145 150 155 160

His Glu Leu Ser His Gly Val Thr Asn Lys Glu Ser Asn Leu Lys Tyr

                165 170 175

Glu Asn Glu Ser Gly Ala Leu Asn Glu Ser Phe Ser Asp Ile Met Gly

            180 185 190

Val Ala Val Glu Gly Lys Asn Phe Val Leu Gly Glu Asp Cys Trp Val

        195 200 205

Ala Gly Gly Val Met Arg Asp Met Glu Asn Pro Ser Arg Gly Gly Gln

    210 215 220

Pro Ala His Met Lys Asp Tyr Lys Tyr Lys Thr Met Asn Asp Asp Asn

225 230 235 240

Gly Gly Val His Thr Asn Ser Gly Ile Ile Asn His Ala Ala Tyr Leu

                245 250 255

Val Ala Asp Gly Ile Glu Lys Thr Gly Ala Lys Asn Ser Lys Asp Ile

            260 265 270

Met Gly Lys Ile Phe Tyr Thr Ala Asn Cys Tyr Lys Trp Asp Glu Thr

        275,280,285

Thr Asn Phe Ala Lys Cys Arg Asn Asp Val Val Gln Val Thr Lys Glu

    290 295 300

Leu Tyr Gly Glu Asn Ser Asn Tyr Val Lys Ile Val Glu Lys Ala Phe

305 310 315 320

Asp Gln Val Gly Ile Thr Ala Thr Pro Gln Leu Pro Leu

                325 330

<210> 2

<211> 581

<212> PROTEIN

<213> Clostridium Botulinum

<400> 2

Met Lys Ser Lys Lys Leu Leu Ala Thr Val Leu Ser Ala Val Ile Thr

1 5 10 15

Phe Ser Thr Val Ser Ala Val Tyr Ala Ala Pro Val Gly Lys Glu Ser

            20 25 30

Lys Val Glu Pro Lys Thr Thr Thr Ile Thr Trp Glu Lys Asn Glu Gln

        35 40 45

Asn Thr Lys Lys Ala Ala Thr Asp Ile Thr Glu Lys Lys Phe Asn Asn

    50 55 60

Ser Glu Glu Ile Thr Lys Phe Phe Glu Lys Asn Ile Ser Lys Phe Gly

65 70 75 80

Val Gln Lys Gly Ser Leu Lys Asn Thr Lys Thr Val Lys Asp Glu Lys

                85 90 95

Gly Lys Thr Asn Tyr His Met Ile Tyr Glu Val Glu Gly Ile Pro Val

            100 105 110

Tyr Tyr Gly Arg Ile Val Phe Thr Thr Glu Lys Asp Ser Ser Met Asp

        115 120 125

Ser Ile Asn Gly Arg Ile Asp Thr Val Phe Glu Asn Gly Asn Trp Lys

    130 135 140

Asn Lys Ile Lys Leu Ser Lys Glu Asp Ala Ile Ala Lys Ala Lys Asn

145 150 155 160

Asp Ile Lys Asp Glu Lys Ala Thr Ser Lys Lys Thr Asp Leu Tyr Leu

                165 170 175

Tyr Asn Phe Glu Gly Lys Pro Tyr Val Val Tyr Leu Val Asp Leu Ile

            180 185 190

Thr Asp Asn Gly Ser Trp Thr Val Phe Val Asn Ala Glu Asp Gly Ser

        195 200 205

Ile Val Asn Lys Phe Asn Asn Thr Pro Thr Leu Ile Asp Thr Lys Asp

    210 215 220

Gln Lys Leu Pro Asn Ala Lys Lys Ile Lys Asp Glu Ala Lys Lys Ala

225 230 235 240

Ser Asn Ala Asn Asn Val Ile Asp Val Gln Gly Gln Ser Val Lys Gly

                245 250 255

Val Gly Lys Thr Ser Leu Asp Gly Leu Val Asn Ile Asp Val Thr Tyr

            260 265 270

Gly Asn Gly Lys Tyr Tyr Leu Lys Asp Ser Asn Lys Asn Ile Tyr Leu

        275,280,285

Tyr Asp Leu Lys Asn Gln Val Asp Glu Tyr Asp Leu Tyr Asn Tyr Leu

    290 295 300

Ser Arg Pro Asn Tyr Lys Gln Ile Leu Met Ser Lys Ser Glu Leu Ile

305 310 315 320

Ser Asn Tyr Asn Asn Asn Phe Ile Ala Asn Asn Gln Val Asn Ser Val

                325 330 335

Asp Ala Tyr Val Asn Thr Asn Lys Thr Tyr Asp Tyr Tyr Lys Asn Lys

            340 345 350

Leu Asn Arg Asn Ser Ile Asp Asn Lys Gly Met Asn Ile Asn Gly Phe

        355 360 365

Val His Val Gly Arg Asn Tyr Gly Asn Ala Phe Trp Tyr Gly Pro Tyr

    370 375 380

Asp Gly Met Phe Phe Gly Asp Gly Asp Gly Ile Tyr Phe Ser Ser Leu

385 390 395 400

Ala Lys Ser Leu Asp Val Val Gly His Glu Leu Ser His Gly Val Thr

                405 410 415

Asn Lys Glu Ser Asn Leu Lys Tyr Glu Asn Glu Ser Gly Ala Leu Asn

            420 425 430

Glu Ser Phe Ser Asp Ile Met Gly Val Ala Val Glu Gly Lys Asn Phe

        435 440 445

Val Leu Gly Glu Asp Cys Trp Val Ala Gly Gly Val Met Arg Asp Met

    450 455 460

Glu Asn Pro Ser Arg Gly Gly Gln Pro Ala His Met Lys Asp Tyr Lys

465 470 475 480

Tyr Lys Thr Met Asn Asp Asp Asn Gly Gly Val His Thr Asn Ser Gly

                485 490 495

Ile Ile Asn His Ala Ala Tyr Leu Val Ala Asp Gly Ile Glu Lys Thr

            500 505 510

Gly Ala Lys Asn Ser Lys Asp Ile Met Gly Lys Ile Phe Tyr Thr Ala

        515 520 525

Asn Cys Tyr Lys Trp Asp Glu Thr Thr Asn Phe Ala Lys Cys Arg Asn

    530 535 540

Asp Val Val Gln Val Thr Lys Glu Leu Tyr Gly Glu Asn Ser Asn Tyr

545 550 555 560

Val Lys Ile Val Glu Lys Ala Phe Asp Gln Val Gly Ile Thr Ala Thr

                565 570 575

Pro Gln Leu Pro Leu

            580

<210> 3

<211> 1296

<212> PROTEIN

<213> Clostridium Botulinum

<400> 3

Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly

1 5 10 15

Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro

            20 25 30

Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg

        35 40 45

Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu

    50 55 60

Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr

65 70 75 80

Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu

                85 90 95

Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val

            100 105 110

Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys

        115 120 125

Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr

    130 135 140

Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile

145 150 155 160

Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr

                165 170 175

Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe

            180 185 190

Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu

        195 200 205

Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu

    210 215 220

Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn

225 230 235 240

Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu

                245 250 255

Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys

            260 265 270

Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn

        275,280,285

Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val

    290 295 300

Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys

305 310 315 320

Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu

                325 330 335

Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp

            340 345 350

Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn

        355 360 365

Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr

    370 375 380

Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn

385 390 395 400

Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu

                405 410 415

Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg

            420 425 430

Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys

        435 440 445

Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe

    450 455 460

Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu

465 470 475 480

Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu

                485 490 495

Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro

            500 505 510

Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu

        515 520 525

Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu

    530 535 540

Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu

545 550 555 560

His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu

                565 570 575

Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys

            580 585 590

Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu

        595 600 605

Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr

    610 615 620

Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala

625 630 635 640

Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu

                645 650 655

Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala

            660 665 670

Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys

        675 680 685

Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu

    690 695,700

Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys

705 710 715 720

Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu

                725 730 735

Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn

            740 745 750

Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp

        755,760,765

Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile

    770 775 780

Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met

785 790 795 800

Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys

                805 810 815

Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly

            820 825 830

Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp

        835 840 845

Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser

    850 855 860

Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn

865 870 875 880

Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser

                885 890 895

Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn

            900 905 910

Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu

        915 920 925

Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser

    930 935 940

Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn

945 950 955 960

Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val

                965 970 975

Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu

            980 985 990

Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser

        995 1000 1005

Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg

    1010 1015 1020

Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln

    1025 1030 1035

Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile

    1040 1045 1050

Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp

    1055 1060 1065

Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu

    1070 1075 1080

Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys

    1085 1090 1095

Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met

    1100 1105 1110

Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Asn

    1115 1120 1125

Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val

    1130 1135 1140

Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr

    1145 1150 1155

Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile

    1160 1165 1170

Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys Asn

    1175 1180 1185

Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu

    1190 1195 1200

Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser

    1205 1210 1215

Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn

    1220 1225 1230

Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly

    1235 1240 1245

Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala

    1250 1255 1260

Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu

    1265 1270 1275

Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu

    1280 1285 1290

Arg Pro Leu

    1295

<210> 4

<211> 25

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / A1

<222> (1) .. (25)

<400> 4

Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly

1 5 10 15

Tyr Asn Lys Ala Leu Asn Asp Leu Cys

            20 25

<210> 5

<211> 25

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / A2 / A6

<222> (1) .. (25)

<400> 5

Cys Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly

1 5 10 15

Tyr Asn Lys Ala Leu Asn Asp Leu Cys

            20 25

<210> 6

<211> 25

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / A3

<222> (1) .. (25)

<400> 6

Cys Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly

1 5 10 15

Tyr Asn Lys Ala Leu Asn Asp Leu Cys

            20 25

<210> 7

<211> 25

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / A3

<222> (1) .. (25)

<400> 7

Cys Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly

1 5 10 15

Tyr Asn Lys Ala Leu Asn Tyr Leu Cys

            20 25

<210> 8

<211> 25

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / A4

<222> (1) .. (25)

<400> 8

Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu Gly

1 5 10 15

Tyr Asn Lys Ala Leu Asn Glu Leu Cys

            20 25

<210> 9

<211> 25

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / A5

<222> (1) .. (25)

<400> 9

Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu Gly

1 5 10 15

Tyr Asn Lys Ala Leu Asn Asp Leu Cys

            20 25

<210> 10

<211> 25

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / A7

<222> (1) .. (25)

<400> 10

Trp Val Arg Gly Ile Ile Pro Phe Lys Pro Lys Ser Leu Asp Glu Gly

1 5 10 15

Ser Asn Lys Ala Leu Asn Asp Leu Cys

            20 25

<210> 11

<211> 10

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / B1 / B4bv / B6

<222> (1) .. (10)

<400> 11

Cys Lys Ser Val Lys Ala Pro Gly Ile Cys

1 5 10

<210> 12

<211> 10

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / B2 / B3

<222> (1) .. (10)

<400> 12

Cys Lys Ser Val Arg Ala Pro Gly Ile Cys

1 5 10

<210> 13

<211> 10

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / B5np

<222> (1) .. (10)

<400> 13

Cys Lys Ser Val Lys Val Pro Gly Ile Cys

1 5 10

<210> 14

<211> 17

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / C / CD

<222> (1) .. (17)

<400> 14

Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn Lys Thr Leu Asp

1 5 10 15

Cys

<210> 15

<211> 14

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / D

<222> (1) .. (14)

<400> 15

Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser Thr Cys

1 5 10

<210> 16

<211> 14

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> BoNT / DC loop

<222> (1) .. (14)

<400> 16

Cys Leu Arg Leu Thr Arg Asn Ser Arg Asp Asp Ser Thr Cys

1 5 10

<210> 17

<211> 15

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / E1-E5

<222> (1) .. (15)

<400> 17

Cys Lys Asn Ile Val Ser Val Lys Gly Ile Arg Lys Ser Ile Cys

1 5 10 15

<210> 18

<211> 15

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / E6

<222> (1) .. (15)

<400> 18

Cys Lys Asn Ile Val Phe Ser Lys Gly Ile Arg Lys Ser Ile Cys

1 5 10 15

<210> 19

<211> 17

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / F1 / F6

<222> (1) .. (17)

<400> 19

Cys Lys Ser Val Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu

1 5 10 15

Cys

<210> 20

<211> 17

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / F2 / F3

<222> (1) .. (17)

<400> 20

Cys Lys Ser Ile Ile Pro Arg Lys Gly Thr Lys Gln Ser Pro Ser Leu

1 5 10 15

Cys

<210> 21

<211> 17

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / F4

<222> (1) .. (17)

<400> 21

Cys Lys Ser Ile Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu

1 5 10 15

Cys

<210> 22

<211> 15

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / F5

<222> (1) .. (15)

<400> 22

Cys Leu Asn Ser Ser Phe Lys Lys Asn Thr Lys Lys Pro Leu Cys

1 5 10 15

<210> 23

<211> 15

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / F7

<222> (1) .. (15)

<400> 23

Cys Lys Ser Ile Val Ser Lys Lys Gly Thr Lys Asn Ser Leu Cys

1 5 10 15

<210> 24

<211> 15

<212> PROTEIN

<213> Clostridium Botulinum

<220>

<221> Loop BoNT / G

<222> (1) .. (15)

<400> 24

Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu Gln Cys

1 5 10 15

<210> 25

<211> 29

<212> PROTEIN

<213> Clostridium Tetani

<220>

<221> TeNT loop

<222> (1) .. (29)

<400> 25

Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn

1 5 10 15

Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys

            20 25

<210> 26

<211> 1005

<212> DNA

<213> C. Botulinum

<400> 26

atggttcaag gtcaaagcgt taaaggagta ggaaaaacta gcttggatgg actagtaaat 60

attgatgtaa cttatggaaa tggaaaatac tatttaaaag atagcaacaa aaatatttat 120

ctatatgact taaaaaatca agttgatgaa tatgatctat acaattatct tagtagacct 180

aactataaac aaatattaat gagcaaatct gaattaatat ctaattacaa taataatttt 240

atagccaaca atcaggttaa ttctgtagat gcttatgtaa acacaaataa aacctatgat 300

tattataaaa acaaattaaa tagaaacagt attgataata agggtatgaa tattaatggg 360

tttgttcatg taggtagaaa ttatggtaat gctttttggt acggtccata tgatgggatg 420

ttctttggcg atggcgacgg aatatacttc tcttcccttg caaaatcttt agatgttgta 480

ggccacgaat taagtcatgg tgtaacaaat aaagagtcta atcttaaata tgaaaatgaa 540

tctggtgccc taaatgaatc tttctcagat attatgggag tagctgttga gggtaaaaac 600

tttgtactag gtgaagattg ctgggttgct ggaggagtaa tgagagatat ggaaaatcca 660

tccagaggag gccaaccagc tcatatgaaa gattataaat acaaaactat gaatgacgat 720

aacggtggtg ttcatacaaa ttcaggtata ataaaccatg ctgcttattt agttgcagat 780

ggaatagaaa aaactggtgc aaaaaatagt aaagatatta tgggaaaaat attctataca 840

gctaattgct ataaatggga tgaaacaaca aattttgcta agtgcagaaa tgatgtagtc 900

caagttacta aagaacttta tggcgaaaat agcaactatg taaaaattgt tgaaaaagct 960

tttgaccaag ttggaataac tgctacacct caattaccat tataa 1005

<210> 27

<211> 1746

<212> DNA

<213> Clostridium Botulinum

<400> 27

atgaaaagta aaaaattatt agctacagtg ctaagtgccg tgatcacttt ttctactgtt 60

tctgcagttt atgctgcgcc tgtaggaaaa gaaagtaaag ttgaaccaaa aactacaaca 120

ataacttggg aaaaaaatga acaaaatact aaaaaagctg ctactgatat aactgaaaag 180

aaatttaaca attctgagga gataactaaa ttctttgaaa aaaatatatc taaatttggt 240

gtacaaaaag gttctcttaa aaacaccaag actgtaaaag acgaaaaagg taaaactaac 300

tatcatatga tttatgaagt agaaggtata cctgtatact atggaagaat tgtttttaca 360

actgaaaaag actcctccat ggattctata aacggtagaa ttgatactgt ttttgaaaat 420

gggaattgga aaaacaaaat caaactatca aaagaagatg ctatagcaaa agctaaaaat 480

gatattaaag atgaaaaagc aactagtaaa aagaccgatt tatatctgta taattttgag 540

ggcaaacctt atgtagttta tttagtagat ctaattacag acaacgggag ttggacggtt 600

ttcgttaatg ctgaggatgg ttctatagta aataaattta ataatactcc tactttaatt 660

gatactaaag atcaaaaatt acccaatgct aaaaaaatta aagatgaagc taaaaaagag 720

agtaatgcaa ataatgtaat tgatgttcaa ggtcaaagcg ttaaaggagt aggaaaaact 780

agcttggatg gactagtaaa tattgatgta acttatggaa atggaaaata ctatttaaaa 840

gatagcaaca aaaatattta tctatatgac ttaaaaaatc aagttgatga atatgatcta 900

tacaattatc ttagtagacc taactataaa caaatattaa tgagcaaatc tgaattaata 960

tctaattaca ataataattt tatagccaac aatcaggtta attctgtaga tgcttatgta 1020

aacacaaata aaacctatga ttattataaa aacaaattaa atagaaacag tattgataat 1080

aagggtatga atattaatgg gtttgttcat gtaggtagaa attatggtaa tgctttttgg 1140

tacggtccat atgatgggat gttctttggc gatggcgacg gaatatactt ctcttccctt 1200

gcaaaatctt tagatgttgt aggccacgaa ttaagtcatg gtgtaacaaa taaagagtct 1260

aatcttaaat atgaaaatga atctggtgcc ctaaatgaat ctttctcaga tattatggga 1320

gtagctgttg agggtaaaaa ctttgtacta ggtgaagatt gctgggttgc tggaggagta 1380

atgagagata tggaaaatcc atccagagga ggccaaccag ctcatatgaa agattataaa 1440

tacaaaacta tgaatgacga taacggtggt gttcatacaa attcaggtat aataaaccat 1500

gctgcttatt tagttgcaga tggaatagaa aaaactggtg caaaaaatag taaagatatt 1560

atgggaaaaa tattctatac agctaattgc tataaatggg atgaaacaac aaattttgct 1620

aagtgcagaa atgatgtagt ccaagttact aaagaacttt atggcgaaaa tagcaactat 1680

gtaaaaattg ttgaaaaagc ttttgaccaa gttggaataa ctgctacacc tcaattacca 1740

ttataa 1746

<---

Claims (16)

1. The use of Lys-C for proteolytic processing of single-stranded botulinum neurotoxin serotype F (BoNT / F) by hydrolysis to form double-stranded botulinum neurotoxin serotype F (BoNT / F), where Lys-C is a Lys-C lysylendopeptidase from Lysobacter enzymogenes . 2. The use of claim 1, wherein the serotype F single chain botulinum neurotoxin (BoNT / F) is a naturally-occurring neurotoxin, recombinant neurotoxin, or a modified neurotoxin, such as a neurotoxin lacking a native H _C domain, or parts thereof, or derivatives with other amino acid residues that replace the H _C domain of a neurotoxin. 3. The use according to claim 2, where the single-stranded botulinum neurotoxin serotype F (BoNT / F) includes an amino acid sequence selected from any of SEQ ID NO: from 19 to 23; preferably where the proteolytically active polypeptide hydrolyzes a single-stranded botulinum neurotoxin of serotype F (BoNT / F) at a position immediately to the C-terminus of the main amino acid residue within the specified sequence from any of SEQ ID NO: 19 to 23. 4. The use according to any one of paragraphs. 1-3, where the C-terminus of the L chain and the N-terminus of the H chain of double-stranded botulinum neurotoxin serotype F (BoNT / F) are identical to the corresponding double-stranded botulinum neurotoxin serotype F (BoNT / F) isolated from wild-type clostridia. 5. The use according to any one of paragraphs. 1-3, where the double-stranded clostridial neurotoxin of serotype F (BoNT / F) has an identical amino acid sequence when compared with the corresponding double-stranded botulinum neurotoxin polypeptide of serotype F (BoNT / F) formed from the same single-stranded botulinum neurotoxin polypeptide of serotype F (BoNT / F) in wild type clostridia. 6. A method of manufacturing a proteolytically processed polypeptide, comprising the step of bringing into contact: (a) a first polypeptide, wherein said first polypeptide is Lys-C, where Lys-C is a lysyl endopeptidase Lys-C from Lysobacter enzymogenes , from (b) a second polypeptide, wherein said second polypeptide is susceptible to proteolysis by said first polypeptide; wherein said contact results in a proteolytic processing of said polypeptide to at least two cleavage products; where the second polypeptide is a single-stranded botulinum neurotoxin of serotype F (BoNT / F) and where said first polypeptide hydrolyzes a single-stranded botulinum neurotoxin of serotype F (BoNT / F) to form a double-stranded botulinum neurotoxin serotype F (BoNT / F). 7. The method of claim 6, wherein said serotype F single chain botulinum neurotoxin (BoNT / F) is a naturally-occurring neurotoxin, recombinant neurotoxin, or a modified neurotoxin, such as a neurotoxin lacking a native H _C domain, or parts thereof, or derivatives thereof other amino acid residues that replace the H _C domain of a neurotoxin. 8. The method according to p. 7, where the second polypeptide includes an amino acid sequence selected from any of SEQ ID NO: from 19 to 23; preferably where the first polypeptide proteolytically cleaves the second polypeptide at a position immediately to the C-end of the main amino acid residue within the specified sequence from any of SEQ ID NO: 19 to 23. 9. The method according to any one of paragraphs. 6-8, wherein said contact occurs in a cell lysate or in a purified cell lysate.

Images