RU2727402C1 - Methods for producing proteolytically processed polypeptides - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, specifically to a proteolytically active polypeptide which is capable of hydrolysing botulinic neurotoxin A (BoNT/A) to produce double-stranded botulinum neurotoxin A (BoNT/A).EFFECT: proteolytically active polypeptide comprising an amino acid sequence having at least 95 % sequence identity of SEQ ID NO: 1 can be used to produce a biologically active double-stranded BoNT/A.11 cl, 11 dwg, 2 tbl, 8 ex

Description

The present invention relates to a novel proteolytically active polypeptide and various uses of the polypeptide in screening and manufacturing 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 interfere with the release of neurotransmitters. Clostridium botulinum secretes seven antigenic variants of botulinum neurotoxin (BoNT) serotypes, designated 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, cleavage participating in the formation of the SNARE complex, 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 the glands. These physiological effects of CNTs on muscle and glandular activity are increasingly being 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 available commercially as a protein formulation of botulinum neurotoxin A, for example under the tradename BOTOX (Allergan Inc.) and under the tradename DYSPORT (Ipsen Ltd.). In a therapeutic application, a complex comprising a 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 formulation without complexing proteins is available under the trade names XEOMIN or Bocouture (Merz Pharmaceuticals GmbH, Frankfurt / Germany). The effect of BoNT is only temporary, which is the reason for the repeated administration of BoNT, which is usually required to maintain the 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 about 150 kDa. Post-translational processing of this single-stranded polypeptide involves limited proteolysis of an overhanging region called a loop (see Table 1) and formation of a nearby disulfide bridge. The active double-stranded neurotoxin consists of two cleavage products resulting from proteolytic hydrolysis of the single-chain 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. 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 that form the catalytic domain and amino acid residues that form the 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 loop region connecting in a single-chain neurotoxin the regions that will become light and heavy chains (cf. Table 1 below).

Purification of CNTs from a fermentation solution is particularly challenging because neurotoxins are contained in it as a mixture of unprocessed, partially processed and fully processed polypeptides that have similar biochemical and physical properties. Usually, partially processed neurotoxins are formed when 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 be formed if endoproteolytic activity has resulted in the detachment 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 the loop peptide can be significantly contaminated from 5% to 90% with partially processed or unprocessed peptides. However, in some cases, the neurotoxin is mostly unprocessed and needs to be treated with endopeptidase before therapeutic use in order to become biologically active.

The prior art describes various attempts to treat Clostridial neurotoxins with heterologous proteases in order to reduce the amount of unprocessed or partially processed precursor proteins. The protease most commonly used to activate Clostridial neurotoxins, trypsin, is useful for activating Clostridial neurotoxins serotypes B (BoNT / B) and E (BoNT / E) (DasGupta & Sugiyama 1972, Biochem. Biophys. Res. Commun. 48: 108- 112; Kozaki et al ., 1974, Infect. Immun. 10: 750-756) is likely to form byproducts, possibly through a proteolytic action near the C-terminus of the heavy subunit BoNT / A, and thus likely interferes with toxin binding with its cellular receptor (Shone et al ., 1985, Eur. J. Bioch. 151: 75-82). The formation of more specific degradation products is theoretically expected from endogenous proteases isolated from a natural host, such as C. botulinum , which produces BoNT / A. Accordingly, various attempts have been made to isolate from the natural host cell endogenous proteases involved in the proteolytic activation of Clostridial neurotoxins. Dekleva and DasGupta (Dekleva & DasGupta, 1989, Biochem. Biophys. Res. Commun. 162: 767-772) were purified from cultures of C. botulinum 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 identified a 62 kDa protein consisting of 15.5 kDa polypeptide and 48 kDa polypeptide. However, the observation of significant CNT fragmentation after the limited exposure of the 62 kDa protein found by Dekleva and DasGupta suggests that the isolated protease may not be the unidentified proteolytic enzyme responsible for CNT activation in Clostridial 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 CNT activation (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), a homologue (74% amino acid identity) of which is present in C. botulinum (CBO1920). C. histolyticum alpha-clostripaine is a cysteine endopeptidase with strong specificity for arginyl bonds. It is synthesized as an inactive preproenzyme that undergoes autocatalytic cleavage to form 15.4 and 43 kDa polypeptides, which combine to form a heterodimeric active enzyme (Dargatz et al. 1993). Both C. histolyticum alpha-clostripain and the C. botulinum 62 kDa protease require a reducing agent and calcium for full activity and are susceptible 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 upregulated by the VirR / VirS two-component system (Shimizu et al. 2002b).

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

Means and methods for reducing the amount of unprocessed and / or partially processed neurotoxin polypeptides and thus improving the quality of neurotoxin preparations are in demand, but still not available. Thus, the technical problem underlying the present invention can be viewed as providing 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 applications 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 to 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 to the sequence of SEQ ID NO: 1. In another aspect, the present invention relates to a proteolytically active polypeptide consisting of a polypeptide sequence as shown in SEQ ID NO: 1.

The term "proteolytically active polypeptide" as used herein refers to the catalytic function of the polypeptide of the present invention and means that the polypeptide of the present invention is capable of hydrolyzing a 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" as used herein refers to the catalytic function of a polypeptide that is the subject of the present invention, and means that the polypeptide, which is the subject of the present invention, is not able to hydrolyze the peptide bond.

One skilled in the art can determine whether a polypeptide, according to the sequence definition described herein, is a polypeptide according to the present invention, by testing the proteolytic activity of the above polypeptide. An assay or test system for determining proteolytic activity comprises contacting a polypeptide that includes a polypeptide sequence having at least 50% sequence identity with SEQ ID NO: 1 with a test substrate. The test substrate is usually a polypeptide known to be cleaved by the polypeptide of the present invention. It is preferred that the test substrate is CNT, such as BoNT, or a fragment thereof. The test substrate can be, for example, uncleaved / unprocessed BoNT, referred to herein as scBoNT, and can be, for example, serotypes A, B, C1, D, E, F, or G (eg, "scBoNT / A", "scBoNT / B ", etc.), or the test substrate may be a tetanus neurotoxin. Alternatively, the test substrate can 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 can be a polypeptide of 50 or more amino acid residues or a peptide up to 49 amino acid residues in length. 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 neurotoxin fragment called LH _N comprising a light chain polypeptide, an overhanging region of a loop peptide and an N-terminal half of a heavy chain polypeptide, an H _N translocation domain. In another aspect, the test substrate is a peptide or includes a peptide selected from any of SEQ ID NOs: 4 to 25 (cf. Table 1). In another aspect, the test substrate is a chimeric neurotoxin comprising amino acid residues derived from two or more serotypes.

A test to determine proteolytic activity will typically include the step of determining the level of conversion of the test substrate to its degradation product (s). Observation of one or more cleavage products formed after contact of the polypeptide with a test substrate, or 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 involve 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 degradation products, and may also include calculating the ratio of substrate to degradation products. In addition, the assay for determining proteolytic activity may include the step of comparing the test sample with a reference sample, wherein the reference sample typically comprises (a) a polypeptide that includes a polypeptide sequence having at least 50% sequence identity with SEQ ID NO: 1, and which is known to have proteolytic activity, and (b) a test substrate known to be cleaved by the polypeptide of (a). In one aspect, the assay for determining proteolytic activity comprises separating the substrate and the degradation 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 labels to aid in detecting a decrease in the amount of the test substrate and / or an increase in the amount of product (s). The term "label" in this context means a detectable marker and includes, for example, a radioactive label, an antibody, a fluorescent label. The amount of test substrate and / or degradation 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 assay for determining the proteolytic activity of a polypeptide of the present invention is described below in the examples illustrating the invention. In a particularly preferred embodiment of the present invention, a polypeptide is proteolytically active if more than 20%, preferably more than 95% of the test substrate is converted to degradation 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-length neurotoxin, but, for example, a fragment of a full-length neurotoxin or a neurotoxin derivative. It is obvious that 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, typically 100 ng of proteolytically active polypeptide and a 1: 100 molar ratio relative to the substrate are used in the assay. In another aspect, a sample can be taken at regular intervals to monitor catalytic activity over time. The assay can be modified, for example, by using different amounts of the 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”, which is 581 amino acid residues long. SEQ ID NO: 1 shows the proteolytically active derivative of SEQ ID NO: 2 lacking amino acid residues at positions 1 to 248 of SEQ ID NO: 2.

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

The term "sequence identity" in this context refers to the determination of identity between the amino acid sequence of comparison and the analyzed sequence, in which the sequences are aligned so as to obtain 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 sequence lengths up to 50 aa. (amino acid) residues, up to 100 aa, up to 150 aa, up to 250 aa, 300 aa, 350 aa, 400 aa, 450 aa, 500 a.k. or 550 a.k. leftovers. In another aspect, sequence identity is calculated for at least 50 aa. residues, at least 100 aa, at least 150 aa. or at least 250 a.k. leftovers. In preferred embodiments of the invention, sequence identity is determined over the entire length of SEQ ID NO: 1 or 2, i.e. for a length of 333 a.k. or 581 a.k., respectively. A series of programs based on different algorithms are available to specialists for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To perform sequence alignment and calculate the sequence identity values listed herein, the commercially available DNASTAR Lasergene MegAlign version 7.1.0 software based on the Clustal W algorithm was used for the entire sequence region with the following settings: paired alignment parameters: sequence skipping penalty: 10.00; gap lengthening penalty: 0.10; 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%, op at least 90%, at least 95% or 100%.

The proteolytically active polypeptide of the present invention may contain the same number of amino acids as the reference polypeptide sequence as shown in SEQ ID NO: 1. Also included in the present invention are polypeptides having additional amino acid residues or fewer amino acid residues. In one aspect, the proteolytically active polypeptide of the present invention is or includes a truncated mutant of SEQ ID NO: 1 or 2, or a polypeptide having at least 50% sequence identity with SEQ ID NO: 1 or 2. In the truncated mutant SEQ ID NO: 2 may, for example, not be one or more amino acid residues located N-terminally from 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 SEQ ID NO: 2 is a C-terminal truncated mutant. In one aspect, the above truncated mutant lacks SEQ ID NO: 2 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 proteolytically active polypeptide of the present invention is at least 200 aa in length. 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 present invention comprises up to 333 aa. 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. leftovers.

In another aspect, the proteolytically active polypeptide of the present invention comprises a polypeptide comprising additional amino acid residues at the N- or C-terminus and / or at an internal position of the polypeptide chain of SEQ ID NO: 1 or a polypeptide sequence having at least 50% identity sequences of SEQ ID NO: 1. These additional amino acid residues can 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 inhibitors of proteolytic activity. In another aspect, additional amino acid residues can be removed by a protease. In another aspect, additional residues that inhibit the proteolytic activity of the polypeptide of the present invention are removed. Additional amino acid residues can 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 to SEQ ID NO: 1 has been modified by replacing one or more amino acid residues. The term "substitution" in this context means the replacement of an amino acid with another amino acid. For example, up to 1 a.c., 2 a.c., 3 a.c., 4 a.c., 5 a.c., 6 a.c., 7 a.c., 8 a.c., 9 aa, 10 aa, 15 aa, 20 aa or up to 50 a.k. can be substituted within the polypeptide sequence. Substitutions can include conservative or non-conservative amino acid substitutions made for the purpose of, for example, increasing or decreasing substrate binding or proteolytic activity of the polypeptide of the present invention.

In one aspect, the proteolytically active polypeptide of the present invention comprises 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 a substrate to two or more cleavage products that are different from the 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 to products derived from the same substrate in 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, the 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-chain neurotoxin is a native double-chain neurotoxin.

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

It should be understood that the definitions and explanations of the terms above and below apply mutatis mutandis to all aspects described in this specification, unless otherwise indicated.

The proteolytically active polypeptide of the present invention is suitable for a variety of applications. A commercially relevant use is in the production of therapeutic neurotoxins such as those isolated from C. botulinum . Currently, C. botulinum cell cultures used to obtain commercially available preparations of botulinum neurotoxin are contaminated with significant amounts of partially processed and / or unprocessed neurotoxin, which have a negative effect, i.e. reduce the specific activity of these pharmaceutical compositions. With the use of the proteolytically active or activated polypeptide of the present invention, for example, after lysis of C. botulinum , it will be possible to treat compositions comprising unprocessed and / or partially processed neurotoxin and thereby convert these contaminating components into fully processed neurotoxin. Consequently, it is possible to make commercial products with increased specific neurotoxin activity, 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 present invention and, in some cases, regulatory elements. The term "regulatory elements" in this context refers to the regulatory elements of gene expression, including transcription and translation, and includes elements such as TATA box, promoter, enhancer, ribosome binding site, Shahin-Dalgarno sequence, internal ribosome entry site (IRES) , a polyadenylation signal, a terminal 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 wild-type cell regulatory element from which a nucleic acid molecule of the present invention is obtained, which is involved in regulating gene expression of a nucleic acid molecule or polypeptide in the above 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 can be, for example, hnRNA, mRNA, RNA, DNA, PNA, LNA and / or modified nucleic acid molecules. The nucleic acid molecule can be cyclic, linear, integrated into the genome, or episomal. Also included are concatemers encoding fusion proteins comprising the three, four, five, six, seven, eight, nine or ten polypeptides of the present invention. Moreover, the nucleic acid molecule may contain sequences encoding signal sequences for intracellular transport, such as signals for transport into an intracellular compartment or for transport across a cell membrane.

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

In another aspect, the present invention relates to a cell containing a nucleic acid molecule or vector of the present invention. The term "cell" as used herein includes prokaryotic and / or eukaryotic cells suitable for expressing the aforementioned nucleic acid molecule or the aforementioned vector, and in particular the polypeptide of the present invention. The above cell may be a host cell not expressing the polypeptide of the present invention, or a homologue thereof. The term "homologue" as used herein refers to a polypeptide comprising a polypeptide sequence having 50% sequence identity with SEQ ID NO: 1. However, the present invention also includes cells, in particular wild-type cells, expressing the polypeptide of the present invention, or its homologue. In a particular aspect, the cell of the present 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, said cell is a C. botulinum Hall strain (ATCC 3502). In another aspect, the above cell is a BoNT / A forming strain ATCC 19397, also known as C. botulinum NCTC 4587 and NCTC 7272. In another aspect, the above cell is a BoNT / A forming C. botulinum strain NCTC 2916. In another aspect, the above cells are BoNT / A2 generating strains Kyoto-F and Mauritius / NCTC 9837 C. botulinum . In another aspect, the above cell is a BoNT / A3 forming strain A254 Loch Maree / NCTC 2012 C. botulinum . In another aspect, the above cell is a BoNT / A4 and B-forming C. botulinum strain CDC657. In another aspect, the above cell is a BoNT / A5 and B3 'generating C. botulinum strain H04402 065. In another aspect, the above cell is a BoNT / B1-forming C. botulinum Okra / NCTC 7273 strain. In another aspect, the above cell is a BoNT / B and F forming C. botulinum CDC4013 / NCTC 12265 strain. In another aspect, the above cell is a BoNT / F1-forming C. botulinum strain Langeland / NCTC 10281. In another aspect, the above cell is a Clostridium sporogenes, Clostridium perfringens, Clostridium acetobutylicum, B. cereus, B. thuringiensis, B. mycoidis, B. thermoproteolyticus, B. anthracis, B. megaterium, B. subtilis, E. coli, or a yeast cell. In one aspect, the polypeptide of the present invention is modified within the cell (ie, glycosylated, phosphorylated, processed by proteases, etc.). The 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 containing an inducer of expression of the polypeptide of the present invention. Such an expression inducer can be a nucleic acid molecule, polypeptide or chemical building block, including small chemical building blocks, having the effect of increasing the amount or activity of the proteolytically active polypeptide of the present invention in cell cultures or lysates thereof. An expression inducer can, for example, increase the transcription or translation of a nucleic acid molecule encoding a polypeptide of the present 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 SEQ ID NO: 2. In one aspect, said cell comprises an inducer , which is a proteolytically active polypeptide capable of removing inhibitory amino acid residues from the N-terminus of the above polypeptide. The inducer can, for example, be expressed by recombinant means known to those skilled in the art. Alternatively, the inducer can be isolated from a cell, for example a clostridial cell.

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

In a related aspect, the present invention provides a method for the production of a proteolytically active polypeptide comprising the steps of: (a) chemically synthesizing or translating from a nucleotide sequence of a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with 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 discussed, 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 a polypeptide of the present invention, compounds other than the above polypeptide. The term also denotes the removal of a polypeptide of the present 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. A variety of 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 rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, or other cell lysates, for example, lysates obtained from C. botulinum and the like. Also included is the translation of the polypeptide of the present invention from the nucleotide sequence of the present invention or from the vector of the present 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 Hall strain C. botulinum (ATCC 3502). Various standard means and methods are available to those skilled in the art for delivering nucleic acid molecules or a vector into a cell and for expressing the polypeptide of the present 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 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). Any of these tools and methods can be used in the methods of the present invention.

The first polypeptide of the present 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 can encode a precursor polypeptide that is proteolytically inactive, but which can be converted to the proteolytically active polypeptide of the present invention. SEQ ID NO: 27 is an example of such a nucleic acid molecule. The proteolytically inactive precursor is also referred to as "inactive BoNT hydrolase", abbreviated as iBH. This proteolytically inactive polypeptide can, for example, be activated during or after translation or upon contact, for example, of the above 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 the polypeptide represented by SEQ ID NO: 2. Another example is a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with SEQ ID NO: 2. The term "inactivating amino acid residues at the N-terminus" in one aspect refers to the first 248 a.k. residues of the above polypeptide. In another aspect, this term refers to a fragment of up to 10 aa, 50 aa, 100 aa, 150 aa, 200 aa, 250 aa. residues of the above polypeptide. Any of these polypeptides are useful in the method of the present 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 aforementioned inactivating amino acid is prepared by producing a fractionated or unfractionated lysate of the aforementioned cells. Inactivating amino acid residues can be removed by contacting the proteolytically inactive polypeptide with the above lysate and incubating until the proteolytically inactive polypeptide is converted to the proteolytically active polypeptide.

In another aspect of the method of the present invention, the polypeptide is translated into a cell. The cell can 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 present invention into 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 above cell is Hall strain C. botulinum (ATCC 3502). In another particular aspect, the above cell is the cell of the present invention as described above.

The translation products obtained by the method of the present invention can be purified by a variety of means that are 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 present invention may include cell lysate centrifugation, ammonium sulfate precipitation of proteins, protein resuspension, centrifugation of 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 purifying the polypeptide of the present invention. A preferred method for purifying a polypeptide of the present invention is described in the Examples, which illustrate the invention.

In one aspect, the purification step comprises linking the polypeptide of the present invention to a solid support. The term "solid support" refers to a matrix including, for example, silicone, cross-linked dextran, cross-linked polyacrylamide, or cross-linked agarose, and the like. Also included are, but are not limited to, 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 can be composed of glass beads and / or polypeptide matrices.

In one aspect, the solid support is associated with an antibody of the present invention. The term "linked" means, in one aspect, stably linked or stably associated. In another aspect, "bound" 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 linked, either directly or via a linker molecule, to a solid support. The antibody can be linked to the above solid support via a linker, including low molecular weight compounds and peptide (or polypeptide) linker molecules. A solid support can actually have any possible structural configuration or organization, provided that it allows the bound antibody to bind to its antigen. Thus, the matrix or solid support can be spherical, as in balls, or cylindrical, as on the inner surface of a test tube or on the outer surface of a rod. Alternatively, the surface can 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 a diagnostic method. In one aspect, the aforementioned manufacturing method may include an affinity chromatography step, wherein the aforementioned affinity chromatography is based on an antibody bound to a solid support. In one embodiment of the present invention, said antibody is an antibody that specifically binds to a proteolytically active polypeptide of the present invention. In another embodiment of the present invention, said antibody is an antibody that specifically binds to a proteolytically inactive polypeptide of the invention.

In another aspect, a method for producing a proteolytically active polypeptide of the present invention comprises purifying the polypeptide of the present invention from a mixture containing additional components. Cleaning can be based on polarity, electrical charge, and size, for example. Thus, the method may in one aspect comprise 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 the chromatographic column can be concentrated, for example, by precipitation or ultrafiltration.

In one aspect, the present invention relates to a composition comprising the proteolytically active polypeptide of the present invention. Using the method disclosed herein, the proteolytically active polypeptide of the present invention can be prepared in which the proteolytically inactive polypeptide is substantially absent. In other words, the method of the present invention provides a proteolytically active polypeptide and a composition free of significant contamination of the inactive precursor protein of the polypeptide of the present invention. It is assumed that the composition does not contain a significant amount of impurities or does not contain a significant amount of proteolytically inactive precursor of the polypeptide if, using the detection method based on Western blot, less than 5% of the proteolytically inactive precursor can be determined, while the above 5% refers to the amount 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 present invention, while the above 50% refers 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 that can be obtained by a method for producing 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 of the polypeptide sequence SEQ ID NO: 1. In another aspect, the proteolytically active polypeptide is a polypeptide having at least 50% sequence identity with the sequence SEQ ID NO: 1. In another aspect, proteolytically the active polypeptide is a polypeptide that includes a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. The term "polypeptide that is obtained" in this context refers in one aspect to a polypeptide that is translated from a nucleic acid that is the subject of the present invention. The polypeptide can be sequentially subjected to post-translational modifications such as acetylation, alkylation, amidation, amino acid addition, amino acid removal, 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 provides, in one aspect, an antibody that specifically binds to a polypeptide of the present invention. The term "antibody" in this context includes a monoclonal antibody, polyclonal antibody, single chain antibody, human, humanized, primatized or chimerized antibody, bispecific antibody, 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 F (ab ') 2, F (ab), Fv, or scFv fragments, or chemically or enzymatically modified derivatives of any of these fragments.

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

In another aspect, the epitope is formed by amino acid residues located N-terminally from amino acid 249 of a polypeptide comprising a polypeptide sequence having at least 50% sequence identity to 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, the epitope to which the antibody of the present invention is specific is an epitope located at the N-terminus of a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with SEQ ID NO: 1. The term “N-terminus "In this aspect of the invention refers to the region 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 polypeptide sequence. In a private aspect, the term refers to 14 N-terminal amino acid residues. The term "epitope" as used herein refers to an antigenic determinant that is recognized by an 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 the proteolytically active polypeptide of the present invention, in which the aforementioned peptide may have a length of 7 to 14, preferably 8, 9, 10, 11, 12, 13 or 14 amino acid residues.

The term “specifically binds” or “specific binding to” in one aspect means that the antibody of the present invention does not substantially cross-react with other epitopes on the polypeptide of the present invention, or other polypeptides in general. Epitope specificity is an important characteristic of the antibody of the present invention. The specificity of an antibody for a proteolytically active polypeptide as compared to a proteolytically inactive polypeptide 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 assays. Another important characteristic is antibody sensitivity. 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 bound sample. Sensitivity can be investigated using well known techniques. Those of skill in the art will be able to determine the operating and optimal test conditions for each determination through routine experimentation. Common binding assay techniques include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, isothermal microcalorimetry, BIACORE® (surface plasmon resonance, SPR) assays, or other surface adsorption techniques. The BIACORE® SPR System measures antigen-antibody interaction. The SPR response reflects the change in mass concentration on the surface of the detector during the binding or dissociation of analytes. Based on SPR, real-time BIACORE® measurements directly track interactions as they occur, see BIAapplications Handbook, version AB (reprinted 1998), BIACORE® code No: BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), BIACORE® code No: BR-1001-84. Binding properties, such as the sensitivity of the antibody of the present invention, can generally be determined in binding assays using an immobilized antigen (ligand) present on the sensor surface. The antibody to be analyzed (analyte) will be present 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 the antibody is injected intermittently across the surface with immobilized antigens, three phases can generally be distinguished: (i) the association of the antibody with the antigen during sample injection; (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 during buffer flow. It should be understood that such a study can alternatively be carried out with immobilized antibodies to be tested and an antigen containing a solution as a mobile phase. The phases of association and dissociation provide information on the kinetics of interactions of the analyte with the ligand (k _a and k _d , rates of complex formation and dissociation, k _d / k _a = K _D ). The equilibrium phase provides information about the affinity of the analyte-ligand interactions (K _D ). In an aspect of the invention, the antibody of the present 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 the 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 produced using the 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 involve the 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 enhance the performance 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 standing from the above epitope. The peptide can be produced, for example, synthetically or by recombinant expression. Alternatively, the antibody of the invention can be produced using the naturally occurring proteolytically active or inactive polypeptide of the invention. In the latter case, it should be understood that the resulting antibodies need to be further tested for specificity to the polypeptide of the present invention. In another aspect of the invention, the monoclonal antibody of the invention is produced using a polypeptide of the invention that can be treated with a detergent to render the epitope immunologically accessible. However, it should be understood that in the event that an antibody is to be directed to a conformational epitope, such treatment with a detergent should not be carried out. In a further aspect, immunostimulating agents such as keyhole limpet 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 the polypeptide of the present invention, as well as for monitoring the presence of the above polypeptide in samples or in recombinant organisms. Moreover, the antibody of the present invention can be used in a detection method or a diagnostic method. In a particular aspect, the antibody of the present 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 the proteolytically active polypeptide of the present invention. Thus, the antibody of the present 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 present invention in a method for the proteolytic processing of a polypeptide. In one aspect, the present invention relates to a method for the production of a proteolytically processed polypeptide, comprising the step of bringing (a) a first polypeptide, said first polypeptide, a polypeptide of the present invention, into contact with (b) a second polypeptide, said second polypeptide, which is subject to proteolysis by 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 arginyl endopeptidase (Arg-C endoprotease, LeR) from Lysobacter enzymogenes (ATCC 29487) (Wright DS, Graham LD, Jennings PA. Biochim Biophys Acta. 1998 Dec 22; 1443 (3): 369-74). Moreover, also included is the use of plasmin and / or omptin (OmpT), a membrane bound serine protease that cleaves (Arg / Lys) - (Arg / Lys) motifs (K. Sugimura and T. Nishihara. J. Bacteriol. 170 ( 1988), pp. 5625-5632) in a CNT proteolytic processing method such as BoNT / A. In one aspect, the present invention provides a method for the production of a proteolytically processed polypeptide, comprising the step of bringing (a) a first polypeptide, said first polypeptide, being Lys-C or Lys-N, into contact with (b) a second polypeptide, said second polypeptide that is subject to proteolysis by said first polypeptide, wherein said contact results in proteolytic processing of said second polypeptide to at least two cleavage products, and in which the second polypeptide is single-chain BoNT / A. The term "Lys-C" refers to the 33 kDa serine endoprotease Lys-C from Lysobacter enzymogenes (lysyl endopeptidase, LeK, Genbank number Q7M135), which specifically cleaves peptide bonds located C-terminally from lysine, or its homologue having at least at least 60% sequence identity. The term "Lys-N" refers to the 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). Also, the term 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 BoNT serotype A, B, C1, D, E, F, or G. Also, the terms "CNT" and "BoNT" includes a recombinant and modified neurotoxin comprising one or more modifications, including chemical modifications or genetic modifications. The term "genetic modification" means the deletion, substitution, or addition of one or more amino acid residues. By using the method of the present invention, it is now possible to prepare neurotoxin compositions with significantly fewer unprocessed or partially processed neurotoxin impurities, since these impurities are efficiently processed to a double-chain 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 allow the physical and / or chemical interaction of the above compounds. According to the method of the present invention, the two different said compounds are, in one aspect, the first and second polypeptides 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" as used herein refers in one aspect to a polypeptide whose polypeptide chain has been hydrolyzed or cleaved at one or more peptide bonds. In another aspect, the term refers to a polypeptide that has been proteolytically cleaved by an endoprotease or endopeptizade. In another aspect, the term refers to a polypeptide that has been cleaved to a level of at least 50%. In another aspect, the above 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. a proteolytically active or activated polypeptide, also referred to as "active BoNT hydrolase". Since the active BoNT hydrolase can be obtained from C. botulinum supernatant, it was originally named native BoNT hydrolase, abbreviated as "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 the substrate of the above first polypeptide. The term "subject to proteolysis" refers to a property or requirement for a second polypeptide and is used herein to mean that a second polypeptide can be proteolytically cleaved by the above first polypeptide. In other words, the term "prone to proteolysis" means that the second polypeptide includes a protease recognition and cleavage site that allows it to act as a substrate for the first polypeptide. The "second polypeptide" is a substrate for the first polypeptide and is proteolytically processed to two or more cleavage products. Using the assay described above, one skilled in the art can check 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 prepare a pharmaceutical composition comprising a Clostridial neurotoxin, or to form polypeptide fragments used in a 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 bringing the first polypeptide into contact with 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 above cell.

In another aspect, the above-mentioned contacting step occurs in a cell lysate or in a purified cell lysate. This aspect includes the addition of the first polypeptide to the lysate or to the 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 protein precipitation, 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 present 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. Also contemplated is the introduction of the first polypeptide to the subject for proteolytic processing of the second polypeptide in the subject. The introduction also includes the joint introduction of the first and second polypeptides. The method also includes the step of incubating under conditions and for a time sufficient to cleave the second polypeptide. In one aspect, conditions may include the addition of 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. The "time sufficient for cleavage" can be determined using the study described above. In one aspect, the aforementioned "time sufficient for cleavage" depends on the level of cleavage that the proteolytically processed polypeptide or composition comprising it must 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 37 ° C. In another aspect, the method includes the step of incubating for up to 1 hour, up to 2 hours, 4 hours, 6 hours, 10 hours, or up to 16 hours.

In one aspect, the polypeptide chain of the above 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 above second polypeptide comprises a sequence selected from any of SEQ ID NO: 4 to 25, and in which the second polypeptide is cleaved C-terminally from a basic amino acid residue within the above sequence from any of SEQ ID NOs: 4 to 25. The above sequences are amino acid sequences of known substrates of the proteolytically active polypeptide of the present invention. Also shown herein is that the above substrates are cleaved towards the C-terminus from the basic amino acid residue contained in the sequence, compare Table 1, columns LC and H _N. In a preferred aspect, said second polypeptide comprises a sequence selected from SEQ ID NO: 4 to 10. In another preferred aspect, said second polypeptide is BoNT / A or a derivative thereof, including, for example, SEQ ID NO: 3 polypeptide and derivatives thereof. 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 truncation of one or more amino acid residues.

In one aspect, the second polypeptide comprises a derivative of any one of SEQ ID NO: 4 to 25 or SEQ ID NO: 3, wherein said derivative contains one or more point mutations and / or one or more additional amino acid residues. In another aspect, the above 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. By using an activity assay to determine protease activity as described herein, one skilled in the art can determine if the above derivative is being processed by the proteolytically active polypeptide of the present invention. In another aspect, the derivative contains a point mutation that changes a basic amino acid residue to a non-basic amino acid residue. In another aspect, the derivative has at least 50% sequence identity to any of SEQ ID NOs: 4 to 25. In another aspect, said 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 above polypeptide comprises (a) a polypeptide sequence having at least 30% sequence identity with SEQ ID NO: 3 [(BoNT / A ATCC 3502, Genbank number AAA23262)]; or (b) a polypeptide sequence selected from the group consisting of tetanus endotoxin, factor X or prothrombin (factor II) blood coagulation cascade protein, pancreatic digestive enzymes such as trypsin, chymotrypsin, pepsin, papain. At least 30% means at least 30%, at least 40%, at least 50%, at least 85%. In a particular aspect, the sequence identity of the aforementioned second polypeptide sequence having at least 50% sequence identity to 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 with a polypeptide sequence located between amino acid positions 420 to 466 of SEQ ID NO: 3 , or at least 30% sequence identity to a polypeptide sequence of any of SEQ ID NOs: 4 to 25. A polypeptide as defined herein may, for example, be obtained from C. botulinum, C. tetani, or C. sporogenes . The above second polypeptide can be, for example, a naturally occurring neurotoxin such as BoNT / A, B, C1, D, E, F or G, or a derivative thereof comprising one or more amino acid mutations such as addition, substitution, deletion or shortening of one or more amino acid residues. Included are, for example, derivatives lacking, for example, the H _C domain of the native neurotoxin or parts thereof, or derivatives with other amino acid residues replacing the H _C domain, as well as derivatives with an additional light chain or other protein cargo 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 at an internal position. Additional amino acid residues can 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), preferably attached to the C-terminus.

In another particular aspect, the above polypeptide is a polypeptide comprising a polypeptide sequence such as shown in GenBank nos CBZ04958.1, YP_002805603.1, ZP_02994746.1, YP_001788403.1, YP_001782792.1, ZP_026.1_16437.1, ZP_026.116437.1, ZP_026.116437.1 1, YP_001255575.1, or a homologue thereof having at least 50% sequence identity.

In another aspect, the biological activity of the above second polypeptide is modulated by proteolytic cleavage. It is well known in the art that the function of many polypeptides can be modulated through proteolytic processing. To "modulate" in this context means to increase or decrease, activate or inactivate. For example, the biological activity of many bacterial neurotoxins is increased or stimulated by the proteolytic processing of a single-chain neurotoxin to a double-chain neurotoxin, in which the double-chain 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 distinct activities: the first activity is the “proteolytic activity” inherent in the light chain of the neurotoxin and is responsible for hydrolysis of the peptide bond of one or more polypeptides involved in the regulation of cell membrane fusion. The second activity is the "translocation activity" inherent in the N-terminus of the heavy chain of the processed neurotoxin and involved in the transport of the light chain across the lysosomal membrane into the cytoplasm. The third activity is the "receptor-binding activity" inherent in the C-terminus of the heavy chain of the processed neurotoxin and is involved in the binding and uptake of the neurotoxin by the target cell. In a preferred aspect, the term "biological activity" in this context means proteolytic activity. In a more preferred aspect, the term means increased proteolytic activity.

The biological activity of the Clostridial neurotoxin can be measured using a variety of tests that are known to those skilled in the art. These tests identify one or more of the activities mentioned above. For example, 50% lethal dose (LD ₅₀ ) study in mice or ex vivo study of the phrenic nerve of the right or left diaphragm dome 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), allow to determine the toxic effect an investigational neurotoxin preparation on a living organism or an isolated neuromuscular preparation. To establish a toxic effect in an LD ₅₀ assay, a neurotoxin must be biologically active and have each of the above activities mentioned above. Moreover, various other studies are available to, for example, determine whether a neurotoxin or a neurotoxin light chain is proteolytically active. Such studies, for example, are based on the contact of BoNT / A with SNAP-25. Alternatively, a peptide representing a cleavage site for SNAP-25 can be used, in which the peptide can be labeled to facilitate detection. In a preferred aspect, biological activity is determined using the MPN assay described below.

In another aspect, said first polypeptide is activated by proteolytic processing of an inactive precursor polypeptide, said inactive precursor polypeptide comprising a polypeptide sequence having at least 60% sequence identity to SEQ ID NO: 2. This aspect is based on the observation that the polypeptide having the polypeptide sequence of SEQ ID NO: 2 is proteolytically inactive, while its N-terminally truncated variants are proteolytically active. The present invention also provides for the use of a proteolytically inactive polypeptide in the methods described herein. A proteolytically inactive polypeptide described herein can be activated, for example, by removing a fragment from the N-terminus or all of the N-terminus comprising 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 auto-proteolysis of SEQ ID NO: 2. 60% sequence identity relates to sequence alignment with respect to full-length NT02CB1447.

In another aspect, the method of the present invention for the production of a proteolytically processed polypeptide comprises the step of purifying the proteolytically processed second polypeptide or at least one or more of its cleavage products. Purification of C. botulinum expressing BoNT / A can be performed, for example, as generally described in the prior art (DasGuppa 1984, Toxicon 22, 415; Sathyamoorthy 1985, J Biol Chemistry 260, 10461). In particular, the purification of the neurotoxin can 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 2% type A NZ-amine medium and the pH is adjusted to 7.2 with 4N NaOH, and then obtained in this way the medium is autoclaved. To this medium, separately autoclaved glucose (30% w / v) can be added to achieve a final glucose concentration in the medium of 0.5%. The incubation can be carried out, for example, at 37 ° C without stirring, while the fermentation is interrupted, for example, after 96 hours. Within the framework of the present invention, in addition to the batch fermentation described previously, it is also possible to carry out semi-continuous fermentation, repeated batch fermentation or continuous fermentation.

After the final fermentation and separation of the fermentation medium from the cells with the fermentation medium, a first precipitation can be carried out to remove large proteins. Preferably, the precipitation is acidic precipitation. The 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 pH 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 again. The precipitate can then be extracted with 0.1 M buffer consisting of citric acid and trisodium citrate, pH 5.5, for example, for an hour. Then you can carry out another centrifugation step, for example, at 9800 × g for 20 minutes at 4 ° C. The resulting precipitate can be extracted again, if necessary, as previously described. The supernatant after extraction and both supernatants, if the extraction is repeated, can then be subjected to protamine sulfate precipitation. The precipitation can continue overnight, for example at 8 ° C. The precipitate can then be centrifuged, for example, for 20 minutes at 4 ° C and 12,000 x g. The supernatant after centrifugation can be subjected to precipitation, such as ammonium sulfate precipitation, during which other large proteins will be removed. After the ammonium sulfate precipitation step, a centrifugation step can be added and then the precipitate thus obtained can be redissolved and, if necessary, dialyzed. With the extract, preferably dialyzed and centrifuged again, a series of chromatographic steps can be carried out in order to purify the neurotoxin. Each chromatographic step serves to remove impurities such as protamine sulfate, residual DNA, portions of smaller and medium-sized proteins, and hemagglutinins from the botulinum neurotoxin protein complex. For this purpose, one or more chromatographic steps can be used in a preferred embodiment of the invention. If necessary, the eluate, for example from the last chromatographic step, can be filtered and suitable adjuvants added. In one aspect, filtration is carried out in reaction containers, which can then undergo a lyophilization step.

The present invention also relates to a composition that can be obtained by the method of the present invention for the production of a proteolytically processed polypeptide. In one aspect, the above composition comprises a mixture of a processed and full length second polypeptide, wherein said mixture may contain less than 5%, 4%, 3%, 2%, or less than 1% of a full length second polypeptide. In an aspect of the above composition, the above second polypeptide is BoNT or a derivative thereof. BoNT can be, for example, selected from the group consisting of BoNT serotypes A, B, C, D, E, F and G, including derivatives thereof. The composition can be, for example, a liquid or solid composition and can 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 a purified double-chain neurotoxin as a drug. In one aspect, said medicament comprises a mixture of processed and full length second polypeptide, wherein said mixture comprises less than 5% full length of second polypeptide. In preferred embodiments of the invention, the mixture contains less than 4%, 3%, 2%, or less than 1% of the unprocessed second polypeptide.

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

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

In another aspect, the present invention relates to a composition according to the present invention for use as a medicament or in a pharmaceutical composition.

In yet another aspect, the present invention relates to an antibody according to the present invention for use as a medicament or in a pharmaceutical composition.

In another aspect, the present invention relates to an inhibitor according to 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 present invention, an antibody of the present invention, a composition of the present invention, or an inhibitor of the present invention.

The term "composition" in this context refers to any composition available in solid, liquid, aerosol (gaseous) form and the like. The above composition comprises, for example, the therapeutically active compound of the present invention, optionally together with suitable adjuvants such as diluents or carriers or other ingredients. In one aspect, the therapeutically active compound is a proteolytically active polypeptide of the present 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 present invention. In another aspect, the therapeutically active compound is an inhibitor of the proteolytically active polypeptide of the present invention.

In this context, for the present invention, a distinction is made between auxiliary compounds, i.e. compounds which do not interfere with 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 the other ingredients. The person skilled in the art can immediately identify such suitable solvents and / or carriers.

The carrier (s) must be compatible with the other ingredients of the formula and must not be harmful to the recipient. The pharmaceutical carrier used may include a solid carrier, a gel, or a 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 wetting agents, and the like. Similarly, the carrier or solvent can 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 others well known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

The solvent (s) are selected so as not to interfere with the biological activity of the combination. Examples of such solvents are distilled water, physiological saline, Ringer's solutions, dextrose solution and Hanks solution, 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 comprises a biologically active neurotoxin prepared by the method of the present invention, optionally one or more pharmaceutically acceptable carriers. The active neurotoxin can be present in liquid or lyophilized form. In one aspect, the above compound may be present together with glycerol, protein stabilizers (eg, human serum albumin (HSA)) or non-protein stabilizers such as polyvinylpyrrolidone or hyaluronic acid. The pharmaceutical composition in one aspect is administered topically. The usually 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 routes. The double-chain neurotoxin polypeptide is the active ingredient of the composition, and in one aspect it is administered in conventional dosage forms prepared by combining the drug with standard pharmaceutical carriers in accordance with conventional procedures. These procedures can include mixing, granulating and compressing or dissolving the ingredients in accordance with the target preparation. It should be understood that the form and nature of the pharmaceutically acceptable carrier or diluent will dictate 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 compound, a neurotoxin, that will be used in a pharmaceutical composition of the present invention that prevents, improves, or treats the symptoms accompanying a 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, 50% effective dose (ED50, 50% efficient dose) (dose therapeutically effective in 50% of the population) and LD ₅₀ (dose lethal for 50% of the population). The dose ratio of therapeutic to toxic effects is a therapeutic index and can be expressed as the ratio LD ₅₀ / ED ₅₀ .

The dosage regimen 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 the patient's weight, body surface area, age, the compound to be administered, gender, time and route of administration, general health, and other drugs that are co-administered. ... Progress can be monitored through periodic assessments. The pharmaceutical compositions and formulas described herein are administered at least once for the purpose of treating or ameliorating or preventing the disease or condition described herein. However, these pharmaceutical compositions can be administered more than once.

In a further aspect of the invention, the above composition is a medicament or a cosmetic composition. In one aspect, the above 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, dystonia of the jaw opening, dystonia of the jaw, bruxism, Meige's syndrome, lingual dystonia of the eyelid, open apraxicia , retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spastic dystonia / adductor type, spastic dystonia / abductor type, spastic dyspnea, limb dystonia, shoulder dystonia, occupational dystonia, writer's spasm MA, musician spasm, golfer spasm, leg dystonia, hip adduction, hip abduction knee flexion, knee extension, foot flexion, foot extension, equinovarus, deformed foot dystonia, spontaneous tonic extension of the big toe, toe flexion, toe extension foot, axial dystonia, "Leaning Tower of Pisa" syndrome, abdominal dystonia, segmental dystonia, unilateral dystonia, generalized dystonia, X-linked dystonia-parkinsonism, dystonia with cortico-basal degeneration, X-linked dystonia-parkinsonism, late dystonia, dystonia spinal-cerebellar ataxia, dystonia with parkinsonism, dystonia with Huntington's chorea, dystonia with Hallevorden-Spatz disease, DOPA-induced dyskinesias / DOPA-induced dystonia, tardive dyskinesias / tardive dystonia, paroxysmal dyskinesias / dystoniae, non-kinesiogenic palate / dystogensis , myoclonic myokymia, rigidity, weak muscle cramps, hereditary chin tremor, paradoxical activity of the masticatory muscles, unilateral spasms of the masticatory muscles, hypertrophic branchial myopathy, hypertrophy of the masticatory muscle, hypertrophy of the anterior tibial muscle, nystagmus, oscillopsy supranuclear paralysis of the glance paralysis, abdominal epilepticus ligaments, refractory mutant dysphoria, upper esophageal sphincter dysfunction, vocal cord granulomas, Gilles de la Tourette's syndrome with stuttering, middle ear myoclonus, protective pharyngeal closure, postlaryngectomy, speech impairment, defensive drooping of the upper eyelid, pseudopion, sphincter dysfunction of Oddi disorders of the esophagus, not associated with achalasia, motor disorders of the esophagus, vaginismus, postoperative immobilization, tremor, bladder dysfunction, detrusor-sphincter dyssynergia, spasm of the urinary bladder sphincter, hemifacial th spasm, reinnervation dyskinesias, wrinkles in the corners of the eyes due to the use of cosmetics, asymmetry of facial muscles, dimples on the chin, muscle stiffness syndrome, tetanus, prostatic hyperplasia, obesity, treatment of strabismus in infantile cerebral palsy, concomitant mixed palsy, condition after surgery for retinal detachment, conditions after cataract surgery, aphakia with myositis strabismus, myopathic strabismus, dissociated vertical displacement, as an adjuvant in strabismus surgery, esotropia, exotropia, achalasia, anal fissures, hyperactivity of exocrine glands, Frey's syndrome, paradoxical lacrimation sweating, axillary, palmar, plantar, 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, Devik's disease, viral infections, bacterial infections, parasitic infections, fungal infections, with hereditary spastic paraparesis postapoplectic syndrome, hemispheric infarction, brain stem infarction, spinal cord infarction, migraine, with trauma of the central nervous system, hemisphere damage, brain stem damage, spinal cord injury brain, hemorrhage in the central nervous system, cerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, intraspinal hemorrhage, with neoplasms, hemispheric tumors, brain stem tumors, spinal cord tumors, snoring (WO 2000/033863). For details and symptoms, see, for example, Jost 2007, Drugs 67 (5), 669 or Dressler 2000 in 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, likewise, it is contemplated that the compound of the present invention is used in one aspect in a substantially pure form. Cosmetic compositions in the following aspect are to be administered 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 comprises an antibody or inhibitor of the present invention. Since the polypeptide of the present invention is responsible for activating 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 infections caused by Clostridia, including Clostridium perfringens, Clostridium difficile, Clostridium tetani, Clostridium botulinum, Clostridium baratii, Clostridium butyricum, Clostridium Clostridium sporogenes, Clostridium acetobium Clostridium novyi and Clostridium oedematiens. Moreover, the antibody of the present invention, in another aspect, can be used to treat symptoms associated with the aforementioned 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 present 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 agent, in particular for treating patients with myocardial infarction, pulmonary embolism, deep vein thromboembolism, i. E. to remove blood clots. Also contemplated is the use of the pharmaceutical composition in the treatment of stroke. 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 surface exposed tumor antigens. 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) contacting a proteolytically active polypeptide of the present invention 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, wherein the reduction in the amount of the cleavage product is indicative of the inhibitory effect of the putative inhibitor. In one aspect, the putative 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 has been replaced by a non-basic amino acid. In a further aspect, the above peptide comprises one or more chemical modifications. In another aspect, the inhibitor is a peptidomimetic of the above peptide. In another aspect, the putative inhibitor is part of a chemical microchip, i. E. groups of organic chemical compounds. In another aspect, the inhibitor is an antibody of the present invention. This method is useful for identifying compounds capable of inhibiting the proteolytically active polypeptide of the present invention. The initial screening can be based, for example, on a peptide comprising an amino acid sequence selected from any of SEQ ID NOs: 4 to 25. Peptides capable of inhibiting a polypeptide of the present invention can be modified to enhance inhibition. Modifications include amino acid substitutions or chemical modifications. Typically, the method is carried out by contacting a polypeptide of the present invention with a known substrate in the presence and absence of a putative inhibitor (step (a) of the method) and by comparing the effect of the putative inhibitor on converting the substrate to a cleavage product. A decrease in the rate of conversion in the presence of a putative inhibitor is indicative of an inhibitory effect.

The present invention also relates to an inhibitor of the proteolytically active polypeptide of the present invention, in which the above inhibitor is (a) an inhibitor comprising an amino acid sequence such as shown in any of SEQ ID NOs: 4 to 25, in which a basic an amino acid has been replaced with a non-basic amino acid; or (b) an antibody of the present invention.

All references cited in this specification are incorporated herein by reference in their entirety disclosing their contents and disclosing the contents specifically mentioned in this specification.

The figures show:

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

5 μl of fractions collected from the HiPrep 16/10 Q FF experiment were analyzed for enzymatic activity by incubating 2 μg scBoNTA (lane 2) for 1 h at 37 ° C followed by 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 by 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. (Lane 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 for determination of protein purity and concentration from three series of nBH purification.

Lane 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 ionization electrospray.

The 38.6 kDa nBH protein band of batch TE311206 was identified as NT02CB1446 / CBO1444 with a Mascot score of 725 and a tandem mass spectrometry peptide sequence coverage of 29.6% over the entire open reading frame (ORF). Not a single peptide was identified (gray rectangle, identified by 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 by tandem mass spectrometry of batch TE311206 showed a sequence coverage of 52% corresponding 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 ionization electrospray.

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

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

A. Electrophoresis in 12.5% SDS-PAGE in an activity assay assaying nBH derived from series TIK301009, TIK280509 and TE311206 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 SDS-PAGE electrophoretic analysis, 3 μl of 4 × Laemmli reducing SDS buffer was added to a final volume of 10 μl. 150 kDa scBoNT / A was cleaved to 100 kDa heavy chain and 50 kDa 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. Higher dilution of the first polypeptide decreases the rate of degradation. The specific proteolytic activity of the 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. In scBoNTAS Throm, all lysine residues are removed and the thrombin recognition sequence LVPRGS is inserted, while in scBoNT Res the loop is devoid of any essential 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. In scBoNTAS CGS-C, the entire loop has been removed and the disulfide bridging cysteines are replaced with glycine and serine.

B. SDS-PAGE electrophoretic analysis of time-dependent cleavage of wild-type and mutant scBoNT / A.

C. scBoNTAS wild type is activated by nBH in a time-dependent manner to 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 cleaved (scBoNTAS Res). Shortening the loop to an 8-mer peptide, introducing five amino acids with bulky side chains, or removing the entire loop leads to the formation of non-cleavable scBoNT / A.

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

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

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

Figure 9: A. Protein content (mg / ml) 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 different lots (500 ng / ml) in phosphate-saline solution overnight at 4 ° C and then blocked for 1 hour with blocking buffer from phosphate-saline solution containing 0 , 1% Tween-20 and 2% skimmed milk. After washing, a dilution of IgY from each pool (10 μg / ml in blocking buffer) was added over 1 h and detected with biotin-labeled donkey immunoglobulins Y against chicken immunoglobulins fused with streptavidin horseradish peroxidase (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 Immobilized Talon Metal Ion Affinity Chromatography (IMAC). Electrophoretic analysis in 10% SDS-PAGE fractions of affinity chromatography on immobilized metal ions Talon (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa; SS34, clarified lysate; TD, unbound fraction; W, wash fraction; E1-E7, imidazole eluted fractions 1 to 7). B. There is no endoproteolysis of scBoNT / A to light chain (50 kDa) and heavy chain (100 kDa) with recombinant iBH (SEQ ID NO: 2, “E”; 63 kDa) at 37 ° C after 1 hour (lane 6) (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa).

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

A. 200 μg of recombinant purified scBoNT / A was incubated with 350 ng of purified active BoNT hydrolase for 12 min at 37 ° C. To stop the reaction, nBH is 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 degradation 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 stop the reaction finally, nBH is 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 degradation 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 stop the reaction finally, nBH is 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 degradation is analyzed by electrophoresis in 10% SDS-PAGE. > 95% processed BoNT / A (SEQ ID NO. 3) is obtained.

The sequence listing shows:

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

SEQ ID NO: 2: Proteolytically inactive polypeptide derived 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: BoNT / A1 loop

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: BoNT / A5 loop

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: Loop BoNT / B5np

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: Loop BoNT / F2 / F3

SEQ ID NO: 21: BoNT / F4 loop

SEQ ID NO: 22: BoNT / F5 loop

SEQ ID NO: 23: BoNT / F7 loop

SEQ ID NO: 24: BoNT / G loop

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 in no way be construed as limiting its scope.

EXAMPLES

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

(1) Readout system / activity study: For the specific detection and purification of enzymatic activity hydrolyzing botulinum neurotoxin A (BoNT / A) 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 the recombinant scBoNT / A with the appropriate enzymatic activity (nBH) should result in 50 kDa LC and 100 kDa HC, visualized by reducing electrophoresis in 10-13% SDS-PAGE.

(2) Expression of Clostridial protease: One colony of C. botulinum strain ATCC 3502 was inoculated into 100 ml of Brain Heart Infusion (BHI) medium and the culture was incubated overnight at 37 ° C. under anaerobic conditions. 10 ml of the overnight culture was inoculated into 1 L of BHI medium and incubated under anaerobic conditions for 48-72 h.

(3) Ammonium sulfate precipitation: 1 L of the 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 xg, 30 min). The precipitated ammonium sulfate precipitate was dissolved in a small volume (here 5 ml) 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 loaded onto an equilibrated 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 performed 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 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 loaded onto a HiLoad 16/60 Superdex 200 column equilibrated with 50 mM NaP pH 7.5, 150 mM NaCl. The separation was carried out at a flow rate of 1 ml / min. Fractions with a retention volume between 80 ml and 100 ml were analyzed by activity assay (1) and appropriate fractions containing enzymatic activity (nBH) were pooled (~ 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). The bound nBH was eluted by decreasing the amount of ammonium sulfate along a linear increasing 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 the activity assay (1) and the appropriate fractions were pooled and concentrated by ultrafiltration to 3.5 ml. The solution buffer was added to 50 mM NaP pH 7.5; 150 mM NaCl.

(7) SEC (HiLoad 16/60 Superdex 75): At the end, 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 migrating at ~ 37.3 kDa were pooled (~ 10 ml) and concentrated to 1 ml by ultrafiltration.

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

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

(1) Tryptic digestion: protein bands of approximately 38 kDa (nBH) in SDS-PAGE electrophoresis were excised for tryptic digestion and discolored by gentle stirring in 50 mM Na ₄ HCO ₃ , 50% acetonitrile for 30 min at 37 ° C. The discoloration was repeated until the spots in the gel became clear. Acetonitrile (100%) was added and removed after 3 minutes. Then the spots were 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 hour. The remaining trypsin solution was then removed, a small volume of 50 mM NH ₄ HCO ₃ was added and digestion was performed at 37 ° C overnight. The supernatant was collected and the gel pieces were extracted using 5% trifluoroacetic acid, 10% acetonitrile twice. 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 MALDI TOF mass spectrometry (Ultraflex 1 Bruker Daltonik GmbH) in a linear mode with an accelerating voltage 25 kV. Masses were detected from 700 m / z to 4500 m / z. Samples (2 μl) were co-crystallized with 2 μl of a sinapic acid solution containing 50% acetonitrile and 0.2% trifluoroacetic acid (TFA) directly on a steel MALDI support. 500 laser pulses were collected for each sample.

(3) Separation of peptides by reversed-phase chromatography: Separation of peptides was performed by reversed-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 onto a C18 column (Zorbax SB-C18, 5 μl, 300 A, 0.5 mm ID, 15 cm length) at flow rate 5 μL / min. After loading, the column was washed for 15 min with buffer A, and 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 min.

(4) Electrospray ionization (ESI ) interface and ion trap mass spectrometry: The HPLC output was directly connected to the nano ESI source of the ion trap mass spectrometer and an Agilent coaxial flow liquid nebulizer (Agilent Technologies) was used. The outlet capillary was held by an adjacent steel needle and protruded 0.1-0.2 mm from it. The nebulization was stabilized with N ₂ as a nebulizer gas (5 L / min). The ionization voltage was set at 4500 V and dry gas was applied at 5 psi (34.47 KPa) and 250 ° C. Spectra were recorded using an Esquire3000 + ion trap mass spectrometer (Bruker Daltonik) at a scan rate of 13000 m / z per second. Using ESI in positive mode, mass spectra were obtained from 50 to 1600 m / z in scan 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 an active exclusion was set for 2 min to exclude precursor ions that were already measured.

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

(5) Results:

table 2
nBH identified by MS Track Party nBH Protein concentration ORF name Genbank number A. to. in ORF Molecular weight [kDa] Mascot indicator 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 score of 725 and a tandem MS peptide sequence coverage of 29.6% throughout the open reading frame (ORF). Not a single peptide derived from the 253 N-terminal amino acids was identified (Figure 4). Analysis of the tandem MS batch TE311206 showed a sequence coverage of 52% corresponding to the C-terminal amino acids 254-594 forming nBH.

37.3 kDa protein bands from lane 3 (nBH batch TIK301009) and lane 4 (nBH batch TIK280509) were identified as NT02CB1447 / CBO1445 with a Mascot score of 555 and 609, respectively. Except for 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% corresponding 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 three purification runs was compared (Figure 6). An activity assay assaying nBH derived from the TIK301009, TIK280509 and TE311206 series using different nBH dilutions shows that higher dilutions decrease the rate of degradation. The proteolytic activity of the three different series is nearly identical, indicating that the mature isoform NT02CB1446 (TE311206) exhibits specific activity similar to the 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 more than 95% of light chain and heavy chain in 120 min. The loop sequence was modified to characterize the cleavage site. In scBoNT / A Throm, all lysine residues are removed and the thrombin recognition sequence LVPRGS is 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 for nBH recognition at the cleavage site. Moreover, loop accessibility of nBH 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) Tandem MS analysis of the 50 kDa scBoNT / A nBH digestion product showed that the C-terminal most assigned peptide covers amino acids G433 through K438, which corresponds to the physiologically observed C-terminus of the scBoNT / A light chain (Figure 8A). Analysis of the 100 kDa cleavage product, which was identified as the BoNT / A heavy chain, showed that the most 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, isolated nBH leads to the formation of physiologically processed BoNT / A and preferably hydrolyzes peptide bonds towards the C-terminus from lysine and arginine residues.

Example 4: Evolutionary Conservation of BoNT Hydrolase and Its Isoforms

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

Sequencing of the C. botulinum ATCC 3502 genome revealed the existence of six ORFs encoding nBH isoforms (Sebaihia et al., 2007, Genome Res. 17 (7): 1082-1092). Subsequent genomic data are available for 10 C. botulinum group I strains, as well as non-BoNT secreting 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: Formation of Antibodies Specific to BoNT Hydrolase

(1) Formation of IgY: Sixteen week old chickens [ISA Brown Lohmann Selected Leghorn (LSL), Spreenhagener Vermehrungsbetrieb fur Legehennen GmbH, Bestensee, Germany] were housed in individual chicken cages specially designed for chickens (Ebeco, Castrop-Rauxel, Germany). Feed (ssniff Leghuhner-Zucht 1 and 2; ssniff Spezialitaten GmbH, Soest, Germany) and water were available without restriction, and the chickens began to lay eggs between 23 and 25 weeks of age. Eggs were harvested daily, labeled, and stored at 4 ° C until later use. All animal care and all experiments were carried out in accordance with the guidelines of the local authorities, Berlin (no. H0069 / 03). Chickens were immunized and boosted intramuscularly (pectoral muscle, left and right sides) 10 times in 1 year at intervals of 4 and 8 weeks. The range used was based on previous work that showed an overt absence of memory cells until 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 in one immunization. Complete Freund's 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, 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 mixing followed by centrifugation (10,000 g for 20 min at 4 ° C), the supernatant was removed and solid PEG 6000 was added to a final concentration of 12% (w / v). This mixture was then 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 electrophoresis in 12.5% SDS-PAGE (Figure 9A) and measured photometrically at 280 nm and calculated according to Lambert-Beer 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 hour blocking buffer from phosphate-saline solution containing 0.1% Tween-20 and 2% skim milk (Merck, Darmstadt, Germany). After washing, a dilution of IgY (10 μg / ml in blocking buffer) was added over 1 h and detected with biotin-labeled donkey IgY against chicken immunoglobulins fused with streptavidin horseradish peroxidase (both from Dianova, Hamburg, Germany) and 3.3 ', 5,5'-tetramethylbenzidine (Sigma). The detected nBH is shown in Figure 9B.

(3) Western blot : nBH was separated by electrophoresis in 12.5% SDS-PAGE and transferred to 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 min and developed using alkaline phosphatase and CDP- Star (Perkin Elmer, Waltham, Massachusetts).

Example 6: Recombinant Expression of BoNT Hydrolase

(1) Plasmid constructs: gene parts encoding native BH (SEQ ID NO: 1) and its propeptide (SEQ ID NO: 2) were amplified by PCR using appropriate oligonucleotides and C. botulinum ATCC 3502 genomic DNA fused to 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 using DNA sequencing.

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

Example 7: Inhibition of BoNT hydrolase

(1) Screening for peptide BH inhibitors: peptides based on SEQ ID NO: 4 to 25 are synthesized without one or more basic residues. Each peptide will be added to the mixture according to the activity assay. A peptide capable of decreasing the amount of processed scBoNT / A, lengthening the time required for complete processing of scBoNT / A, or blocking processing of scBoNT / A is considered an nBH inhibitor.

(2) Screening of antibody-based inhibitors: Antibodies raised against epitopes derived from nBH like the IgY of Example 5 are incubated with nBH and subsequently assayed for activity. An antibody capable of decreasing 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.

Example 8: Use of Purified Active BoNT Hydrolase (nBH) to Produce 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 is 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 performed 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 stop the reaction finally, the nBH is 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 performed 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 stop the reaction finally, the nBH is 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 performed by electrophoresis in 10% SDS-PAGE (Figure 11C). > 95% processed BoNT / A (SEQ ID NO: 3) is obtained. 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 incubated with 350 ng nBH). After incubation for 1 h at 37 ° C, more than 97% of the BoNT / A is processed.

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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 Val

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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> BoNT / C / CD hinge

<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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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> Hinge 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 hinge

<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 taaaaaagct 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 (19)

1. A proteolytically active polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO ID: 1, wherein said proteolytically active polypeptide is capable of hydrolyzing botulinum neurotoxin A (BoNT / A) to produce double-stranded botulinum neurotoxin A (BoNT / A ). 2. A nucleic acid molecule encoding a proteolytically active polypeptide according to claim 1. 3. A vector for expression of a proteolytically active polypeptide according to claim 1, wherein said vector contains a nucleic acid molecule according to claim 2 and regulatory elements. 4. A cell for producing a proteolytically active polypeptide according to claim 1, wherein said cell contains a nucleic acid molecule according to claim 2 or a vector according to claim 3. 5. A method for the production of a proteolytically active polypeptide according to claim 1, comprising the steps of: (a) chemical synthesis or translation of a polypeptide from a nucleotide sequence, wherein said polypeptide consists of a proteolytically active polypeptide according to claim 1; and (b) purifying the polypeptide obtained in step (a). 6. The use of a proteolytically active polypeptide according to claim 1 for producing an antibody that specifically binds to said proteolytically active polypeptide. 7. A method for the production of a proteolytically processed polypeptide, including the stage of bringing into contact: (a) a first polypeptide, wherein said first polypeptide comprises a proteolytically active polypeptide according to claim 1, with (b) a second polypeptide, wherein said second polypeptide comprises a sequence having at least 95% identity to any of SEQ ID NO: 3-5, wherein said contact results in proteolytic processing of said second polypeptide to at least two cleavage products. 8. The method of claim 7, wherein the second polypeptide is cleaved at a position immediately C-terminus from the basic amino acid residue within said sequence selected from any of SEQ ID NO: 3-5. 9. A method for screening an inhibitor of a proteolytically active polypeptide according to claim 1, comprising the steps of: (a) contacting the proteolytically active polypeptide of claim 1 with a single-chain botulinum neurotoxin A (BoNT / A) and a putative inhibitor; and (b) determining the effect of a putative inhibitor on the conversion of single-chain botulinum neurotoxin A (BoNT / A) to double-chain botulinum neurotoxin A (BoNT / A), wherein the reduction in the amount of double-chain botulinum neurotoxin A (BoNT / A) is indicative of the inhibitory effect of the putative inhibitor. 10. The use of a proteolytically active polypeptide according to claim 1 in the preparation of a therapeutic double-chain botulinum neurotoxin A (BoNT / A). 11. A composition for hydrolyzing botulinum neurotoxin A (BoNT / A) to obtain a double-chain botulinum neurotoxin A (BoNT / A) containing the proteolytically active polypeptide of claim 1 in an effective amount and a carrier.

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