RU2782382C2 - Hybrid neurotoxins - Google Patents

Abstract

FIELD: biotechnology; biochemistry.

SUBSTANCE: invention relates to the field of biotechnology and biochemistry, specifically to hybrid polypeptide for binding to ganglioside. Hybrid polypeptide contains a light chain of clostridium neurotoxin (L), HN domain of clostridium neurotoxin, HCN domain of clostridium neurotoxin, HCC domain of clostridium neurotoxin with amino acid sequences at least 95% identical to sequences with amino acid residues, respectively, 1-448 SEQ ID NO: 1, 449-872 SEQ ID NO: 1, 873-1094 SEQ ID NO: 1, 1095-1296 SEQ ID NO: 1; and a selective ganglioside-binding fragment capable of binding to GM1, with an amino acid sequence at least 95% identical to a sequence with amino acid residues 22-124 SEQ ID NO: 9. The invention also relates to a nucleotide sequence encoding the specified hybrid polypeptide, an expression vector containing the specified sequence, and a cell for expression of the specified hybrid polypeptide, containing the specified vector.

EFFECT: invention allows for increase in selectivity of affinity of ganglioside binding.

11 cl, 6 dwg, 1 tbl, 2 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to hybrid neurotoxins with improved therapeutic properties, in particular with more selective ganglioside binding affinity.

BACKGROUND OF THE INVENTION

Bacteria of the genus Clostridia produce highly potent and specific protein toxins that can poison neurons and other cells they enter. Examples of such Clostridial toxins include neurotoxins produced by C. tetani (TeNT) and C. botulinum (BoNT) AG serotypes, as well as those produced by C. baratii and C. butyricum .

Clostridial neurotoxins contain some of the most potent toxins known. For example, botulinum neurotoxins have a median lethal dose (LD50) in mice ranging from 0.5 to 5 ng/kg, depending on serotype. Both tetanus and botulinum toxins act by inhibiting the function of the affected neurons, in particular the release of neurotransmitters. While botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system.

In nature, Clostridial neurotoxins are synthesized as a single-chain polypeptide, which is post-translationally modified as a result of proteolytic cleavage to form two polypeptide chains connected to each other by a disulfide bond. The cleavage occurs at a specific cleavage site, often referred to as the activation site, which is located between the cysteine residues providing the interchain disulfide bond. It is this double-stranded form that is the active form of the toxin. The two chains are called the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain includes an N-terminal translocation component (H _N -domain) and a _C -terminal guide component (HC-domain). The cleavage site is located between the L chain and the translocation domain. After binding the HC domain to its target neuron and internalizing the bound toxin into the cell via the endosome, the H _N domain translocates the L chain across the _endosomal membrane and into the cytosol, and the L chain provides a proteolytic function (also known as a non-cytotoxic protease).

Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin) - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE is derived from the term "soluble attachment receptor NSF", where NSF stands for N-ethylmaleimide sensitive factor. SNARE proteins are required for the fusion of intracellular vesicles and thus for the secretion of molecules via vesicular transport out of the cell. The proteolytic function is a zinc-dependent endopeptidase activity and has a high substrate specificity for SNARE proteins. Accordingly, once delivered to the desired target cell, the non-cytotoxic protease is able to inhibit cellular secretion from the target cell. The proteolytic L-chains of Clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins. Proteolytic L chains from BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave VAMP, proteolytic L chains from BoNT/A and BoNT/E cleave SNAP25, and proteolytic L chain BoNT/C cleaves both SNAP25, and syntaxin, leading to inhibition of neurotransmitter release and subsequent neuroparalysis (Rossetto, O. et al., "Botulinum neurotoxins: genetic, structural and mechanistic insights." Nature Reviews Microbiology 12.8 (2014): 535-549).

Because SNARE proteins are expressed in all cells, Clostridial neurotoxins such as botulinum toxin have been successfully used in a wide range of therapies. Currently, all approved medicines/cosmetics containing BoNT contain naturally occurring neurotoxins purified from Clostridial strains (BoNT/A for DYSPORT®, BOTOX® or XEOMIN®, and BoNT/B for MYOBLOC®).

As an example, the inventors refer to William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004), which describes the use of Clostridium neurotoxins such as botulinum neurotoxins (BoNT), BoNT/A, BoNT/ B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, as well as tetanus neurotoxin (TeNT), to inhibit neuronal signaling in a range of therapeutic and cosmetic or aesthetic applications - e.g. commercially available botulinum toxin products are currently approved as therapeutic agents for indications including focal spasticity, upper limb spasticity, lower limb spasticity, cervical dystonia, blepharospasm, hemifacial spasm, axillary hyperhidrosis, chronic migraine, neurogenic detrusor overactivity, glabellar wrinkles, and deep lateral periorbital wrinkles. In addition, Clostridial neurotoxin therapy has been described for the treatment of neuromuscular disorders (see US 6,872,397); for the treatment of diseases of the uterus (see US 2004/0175399); for the treatment of peptic ulcer and gastroesophageal reflux disease (see US 2004/0086531); for the treatment of dystonia (see US 6319505); for the treatment of eye diseases (see US 2004/0234532); for the treatment of blepharospasm (see US 2004/0151740); for the treatment of strabismus (see US 2004/0126396); for the treatment of pain (see US 6869610, US 6641820, US 6464986 and US 6113915); for the treatment of fibromyalgia (see US 6623742, US 2004/0062776); for the treatment of low back pain (see US 2004/0037852); for the treatment of muscle injuries (see US 6423319); for the treatment of headache in the paranasal sinuses (see US 6838434); for the treatment of tension headache (see US 677692); for the treatment of headache (see US 6458365); to reduce migraine headache (see US 5714469); for the treatment of cardiovascular diseases (see US 6767544); for the treatment of neurological diseases such as Parkinson's disease (see US 6620415, US 6306403); for the treatment of neuropsychiatric diseases (see US 2004/0180061, US 2003/0211121); for the treatment of endocrine diseases (see US 6827931); for the treatment of diseases of the thyroid gland (see US 6740321); for the treatment of cholinergic diseases of the sweat glands (see US 6683049); for the treatment of diabetes (see US 6337075, US 6416765); for the treatment of diseases of the pancreas (see US 6261572, US 6143306); for the treatment of cancers such as bone tumors (see US 6565870, US 6368605, US 6139845, US 2005/0031648); for the treatment of ear diseases (see US 6358926, US 6265379); for the treatment of autonomic disorders such as diseases of the gastrointestinal muscles and other disorders of the functions of smooth muscles (see US 5437291); for the treatment of skin lesions associated with proliferative diseases of skin cells (see US 5670484); for the treatment of neurogenic inflammatory diseases (see US 6063768); to reduce hair loss and stimulate hair growth (see US 6299893); for the treatment of lowered corners of the mouth (see US 6358917); to reduce appetite (see US 2004/40253274); in dental therapy and procedures (see US 2004/0115139); for the treatment of neuromuscular diseases and disorders (see US 2002/0010138); for the treatment of various diseases and disorders and associated pain (see US 2004/0013692); for the treatment of conditions resulting from increased mucus secretion such as asthma and COPD (see WO 00/10598); and for the treatment of non-neuronal diseases such as inflammation, endocrine diseases, exocrine disorders, immunological diseases, cardiovascular diseases, bone diseases (see WO 01/21213). All of the above publications are incorporated herein by reference in their entirety.

However, native BoNTs do not discriminate between the spatial distribution of neuromuscular junctions or different types of neurons, which can lead to side effects. For example, treatment of upper limb spasticity with BoNT can lead to adverse events such as dry mouth and dysphagia (Nair, K. P., and Jonathan Marsden. "The management of spasticity in adults." Bmj 349 (2014): g4737).

Increasing the specificity of Clostridial neurotoxins to certain populations of neurons will allow the adaptation of Clostridial neurotoxin to certain pathologies with increased safety and reduced side effects.

The present invention relates to neurotoxins with improved therapeutic properties, in particular with a more selective binding affinity for certain neurons providing muscle contraction (neuromuscular junction) or cholinergic secretion.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a hybrid neurotoxin comprising a Clostridial neurotoxin light chain (L) and a selective ganglioside binding moiety (GBM), wherein the selective ganglioside binding moiety is not a _{Clostridia HCC} or HC domain.

In another aspect, the present invention provides a nucleotide sequence encoding a hybrid neurotoxin of the invention.

In another aspect, the present invention relates to a vector containing the nucleotide sequence of the invention.

In another aspect, the present invention relates to a cell containing the nucleotide sequence or vector of the invention.

In another aspect, the present invention relates to a pharmaceutical composition containing a hybrid neurotoxin according to the invention.

In another aspect, the present invention relates to a hybrid neurotoxin or pharmaceutical composition of the invention for use in therapy.

In another aspect, the present invention relates to the non-therapeutic use of a hybrid neurotoxin or pharmaceutical composition of the invention for the treatment of an aesthetic or cosmetic disorder.

In another aspect, the present invention provides a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a _{Clostridia HCC} or HC domain, and wherein said selective ganglioside binding moiety binds to one or more gangliosides selected of GM1a, GM1b, GD1a and GalNAc-GD1a, for use in the treatment of limb disorders associated with unwanted neuronal activity.

In another aspect, the present invention provides a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a _{Clostridia HCC} or HC domain, and wherein said selective ganglioside binding moiety binds to one or more gangliosides selected of GT1a and GQ1b, for use in the treatment of diseases of the head or neck associated with unwanted neuronal activity.

In another aspect, the present invention provides a method of treatment comprising administering a therapeutically effective amount of a hybrid neurotoxin or pharmaceutical composition of the invention to a patient in need thereof.

In another aspect, the invention provides a hybrid neurotoxin comprising a clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a Clostridia HCC or _HC domain, and wherein said selective ganglioside binding moiety binds to GM1 for use in the treatment of _sialorrhea ( or excessive salivation or drooling).

In another aspect, the invention provides a hybrid neurotoxin comprising a clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a _{Clostridia HCC} or HC domain, and wherein said selective ganglioside binding moiety binds to one or more gangliosides selected from NeuAc GM3, NeuGc GM3, GM2, GM1, GD3 and GD2, for use in cancer treatment.

In another aspect, the present invention relates to a method of treating a limb disease associated with unwanted neuronal activity comprising administering to a patient in need thereof a therapeutically effective amount of a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not _HCC - or H _C -domain of Clostridium, and where the specified selective ganglioside-binding fragment binds to one or more gangliosides selected from GM1a, GM1b, GD1a and GalNAc-GD1a.

In another aspect, the present invention provides a method of treating a head or neck disease associated with unwanted neuronal activity, comprising administering to a patient in need thereof a therapeutically effective amount of a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not H _CC - or H _C -domain of Clostridia, and where the specified selective ganglioside-binding fragment binds to one or more gangliosides selected from GT1a and GQ1b.

In another aspect, the present invention relates to a method of treating sialorrhea (or excessive salivation or drooling) comprising administering to a patient in need thereof a therapeutically effective amount of a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not _HCC - or H _C -domain of Clostridia, and where the specified selective ganglioside-binding fragment binds to GM1.

In another aspect, the present invention relates to a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a _{Clostridial HCC} or HC domain, and where the specified selective ganglioside-binding fragment binds to one or more gangliosides selected from NeuAc GM3, NeuGc GM3, GM2, GM1, GD3 and GD2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the inventors' discovery that the neuromuscular selectivity of Clostridial neurotoxin can be altered by introducing exogenous (non-clostridial) ganglioside-binding domains into Clostridial neurotoxin.

Botulinum neurotoxins (BoNT) interact with neurons using a dual receptor binding mechanism involving protein receptors and plasma membrane gangliosides. BoNT/B/G and /DC have been shown to recognize the luminal domain of SytI and SytII (two major isoforms of synaptotagmin). Synaptic vesicular glycoproteins 2 (SV2), including the three isoforms SV2A, SV2B, and SV2C, have been shown to be protein receptors for BoNT/A, BoNT/D, BoNT/E, BoNT/F.

Gangliosides are oligoglycosylceramides derived from lactosylceramide and containing a sialic acid residue such as N-acetylneuraminic acid ("NANA", "SA", "Neu5Ac" or "NeuAc"). In some cases, the sialic acid component is N-glycolylneuraminic acid (Neu5Gc) or a Neu5Ac analog in which the amino group is replaced by OH (3-deoxy-D-glycero-D-galactononulosonic acid, abbreviated as "KDN"). Gangliosides are classified according to the nomenclature system proposed by Svennerholm, in which M, D, T, and Q refer to mono-, di-, tri-, and tetrasialogangliosides, respectively, and the numbers 1, 2, 3, etc. refer to the migration order of gangliosides in thin layer chromatography. For example, the migration order of monosialogangliosides is GM3 > GM2 > GM1. Additional terms are added to indicate differences in basic structures, such as GM1a, GD1b, etc. Glycosphingolipids containing 0, 1, 2, and 3 sialic acid residues linked to an internal galactose unit are called asialo (or 0-), a-, b-, and c-series gangliosides, respectively, while gangliosides having sialic acid residues associated with an internal N-galactosamine residue are classified as α-series gangliosides. Ganglioside biosynthetic pathways of series 0, a, b, and c involve sequential activity of sialyltransferases and glycosyltransferases, as illustrated, for example, in Ledeen et al., 2015 (Ledeen et al., 2015 (Ledeen, Robert W., and Gusheng Wu. "The multi-tasked life of GM1 ganglioside, a true factotum of nature." Trends in biochemical sciences 40.7 (2015): 407-418). and a heterogeneous spectrum of products such as α-series gangliosides with sialic acid moiety(s) linked to an internal N-acetylgalactosamine moiety.

Gangliosides are transported to the outer side of the plasma membrane by a transport system involving the formation of vesicles. Gangliosides are present and concentrated on the cell surface, with the two hydrocarbon chains of the ceramide moiety embedded in the plasma membrane, and the oligosaccharides are on the extracellular surface where they represent recognition sites for extracellular molecules or neighboring cell surfaces. The sialoglican components of gangliosides protrude above the cell surface, where they can participate in intermolecular interactions. They function by recognizing specific molecules on the cell surface and by regulating the activity of proteins at the plasma membrane. Gangliosides also bind specifically to viruses and bacterial toxins such as botulinum, tetanus, and cholera toxins. For example, a specific cell surface receptor for cholera toxin is the GM1 (or GM1a) ganglioside: Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer.

_BoNTs have two independent binding regions for gangliosides and neuronal protein receptors in the HCC domain. BoNT/A/B/E/F and /G have a conserved ganglioside-binding site in the H _CC domain consisting of the "E(Q) ... H (K) ... SXWY ... G" motif, while BoNT/C/D and / DCs have two independent ganglioside binding sites. (Lam, Kwok-Ho, et al. "Diverse binding modes, same goal: The receptor recognition mechanism of botulinum neurotoxin." Progress in biophysics and molecular biology 117.2 (2015): 225-231.) Most BoNTs bind only to gangliosides, which have a 2,3-linked N-acetylneuraminic acid residue (denoted Sia5) attached to Gal4 of the oligosaccharide core, while the corresponding ganglioside-binding pocket on TeNT can also bind to GM1a, a ganglioside that lacks a Sia5 sugar residue. Introduction of the H1241K mutation into recombinant BoNT/F has been shown to confer GM1 binding capability (Benson, Marc A., et al. "Unique ganglioside recognition strategies for clostridial neurotoxins." Journal of Biological Chemistry 286.39 (2011): 34015-34022). BoNT/D has been found to bind GM1a and GD1a (Kroken, Abby R., et al. "Novel ganglioside-mediated entry of botulinum neurotoxin serotype D into neurons." Journal of Biological Chemistry 286.30 (2011): 26828-26837).

Combining data from ganglioside-deficient mice and from biochemical assays, BoNT/A, E, F, and G preferentially bind to the NAcGal-Gal-NAcNeu terminal fragment present in GD1a and GT1b, while BoNT/B , C, D and TeNT require a disialyl motif present in GD1b, GT1b and GQ1b. Thus, it appears that common complex polysialogangliosides such as GD1a, GD1b, and GT1b are required for the specific accumulation of all BoNT and TeNT serotypes on the surface of neuronal cells as the first stage of intoxication. (Rummel, Andreas. "Double receptor anchorage of botulinum neurotoxins accounts for their exquisite neurospecificity." Botulinum Neurotoxins. Springer Berlin Heidelberg, 2012. 61-90.)

The inventors hypothesized that one could take advantage of the differential localization of gangliosides in the body to increase the selectivity of Clostridial neurotoxins for neurons at certain positions. The inventors, in particular, have shown that the B-subunit of cholera toxin can be used to transfer the ability to bind GM1 to BoNT/A.

In a first aspect, the present invention relates to a hybrid neurotoxin comprising a Clostridial neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a Clostridial H _C C or H _C domain.

As used herein, the term "neurotoxin" means any polypeptide that enters a neuron and inhibits the release of a neurotransmitter. This process includes binding of the neurotoxin to a low-affinity or high-affinity receptor, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin into the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term "neurotoxin" encompasses any polypeptide produced by Clostridium bacteria ( Clostridial neurotoxins) that enters a neuron and inhibits the release of a neurotransmitter, and such polypeptides obtained by recombinant techniques or chemical methods. This double-stranded form is the active form of the toxin. The two chains are called the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa.

Different serotypes of botulinum neurotoxin (BoNT) can be distinguished by inactivation by specific neutralizing antisera, and this serotype classification correlates with percent sequence identity at the amino acid level. The BoNT proteins of a given serotype are further divided into different subtypes based on percent amino acid sequence identity. An exemplary BoNT/A amino acid sequence is provided as SEQ ID NO: 1 (UniProt database accession number A5HZZ9). An exemplary BoNT/B amino acid sequence is provided as SEQ ID NO: 2 (UniProt accession number B1INP5). An exemplary BoNT/C amino acid sequence is provided as SEQ ID NO: 3 (UniProt accession number P18640). An exemplary BoNT/D amino acid sequence is provided as SEQ ID NO: 4 (UniProt accession number P19321). An exemplary BoNT/E amino acid sequence is provided as SEQ ID NO: 5 (UniProt database accession number Q00496). An exemplary BoNT/F amino acid sequence is provided as SEQ ID NO: 6 (UniProt database accession number Q57236). An exemplary BoNT/G amino acid sequence is provided as SEQ ID NO: 7 (UniProt database accession number Q60393). An exemplary amino acid sequence of TeNT (tetanus neurotoxin) is shown as SEQ ID NO: 8 (UniProt accession number P04958).

As used herein, the term "clostridia neurotoxin light chain" (or "L") refers to a Clostridial endopeptidase domain (or non-cytotoxic protease) of approximately 50 kDa that has the ability to cleave the SNARE protein and thereby impair neurotransmitter release from the target cell.

As used herein, the term " _HN -domain" refers to a functionally distinct neurotoxin heavy chain region of approximately 50 kDa that has the ability to translocate the Clostridial neurotoxin light chain into the cytoplasm of a target cell.

As used herein, the term "LH _N -domain" means a neurotoxin that lacks an _HC -domain and consists of an endopeptidase domain ("L" or "light chain") and a domain responsible for translocation of endopeptidase into the cytoplasm ( _HN -domain of heavy chains). The LH _N domain contains an activation site between the L domain and the H _N domain. In proteolytic cleavage of the activation site, the L-domain and _HN -domain are joined together by a disulfide bond.

As used herein, the term " _HC domain" refers to a functionally distinct region of the heavy chain of a neurotoxin with a molecular weight of approximately 50 kDa, which allows the neurotoxin to bind to a receptor located on the surface of a target cell. The HC domain consists of two structurally distinct subdomains, the "H _CN subdomain" (N-terminal portion of the HC domain) and the "H _CC subdomain" ( _C -terminal portion of the HC domain ₎ , each of which has a molecular weight of approximately 25 kDa.

Examples of L, H _N , H _CN and H _CC domains are shown in Table 1.

Table 1 - Examples of domains L, H _N , H _CN and H _CC .

Neurotoxin Access number SEQID NO L H _N H _CN H _CC BoNT/A1 A5HZZ9 one 1-448 449-872 873-1094 1095-1296 BoNT/B1 B1INP5 2 1-441 442-859 860-1081 1082-1291 BoNT/C1 P18640 3 1-449 450-867 868-1095 1096-1291 BoNT/D P19321 four 1-442 443-863 864-1082 1083-1276 BoNT/E1 WP_003372387 5 1-423 424-846 847-1069 1070-1252 BoNT/F1 Q57236 6 1-439 440-865 866-1087 1088-1278 BoNT/G WP_039635782 7 1-446 447-864 865-1089 1090-1297 TeNT P04958 eight 1-456 457-880 881-1111 1112-1315

The above reference sequences should be considered as an example, since slight differences may be observed depending on the subserotypes. By way of example, US 2007/0166332 (thus incorporated by reference in its entirety) provides slightly different sequences from Clostridia.

In one embodiment, the Clostridial neurotoxin light chain is BoNT type A, type B, type C1, type D, type E, type F, or type G, or TeNT.

In one embodiment, the Clostridial neurotoxin light chain domain contains a sequence selected from:

- amino acid residues 1 to 448 of SEQ ID NO: 1 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1 to 441 of SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1 to 449 of SEQ ID NO: 3 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1 to 442 of SEQ ID NO: 4 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1 to 423 of SEQ ID NO: 5 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1 to 439 of SEQ ID NO: 6 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1 to 446 of SEQ ID NO: 7 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them, and

- amino acid residues 1 to 456 of SEQ ID NO: 8 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them.

It is understood that the Clostridial neurotoxin light chain of the invention has the ability to cleave the SNARE protein.

In one embodiment, the hybrid neurotoxin contains a translocation fragment.

The term "translocating fragment" (or "translocating domain"), as used herein, means a group that has the ability to transfer (translocate) the light chain of Clostridium neurotoxin into the cytoplasm of the target cell.

Suitable translocation fragments include bacterial toxin translocation domains such as Clostridia HN domains and/or the cholera toxin A2 subunit ( _CtxA2 ), cell-penetrating peptides, in particular pH sensitive cell-penetrating peptides. An example of a pH-sensitive cell-penetrating peptide is HBHAc (KKAAPAKKAAAKKAPAKKAAAKK), including a pH-sensitive masking peptide, histidine glutamic acid (HE) (Yeh et al, Mol Pharm 2016 ʺSelective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.ʺ).

In a preferred embodiment, the hybrid neurotoxin contains a translocation fragment that is the H _N -domain of Clostridia. More preferably, the hybrid neurotoxin contains an activation site between the light chain and the H _N -domain of Clostridia.

In one embodiment, the H _N -domain of Clostridia contains a sequence selected from:

- amino acid residues 449 to 872 of SEQ ID NO: 1 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 442 to 859 of SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 450 to 867 of SEQ ID NO: 3 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 442 to 863 of SEQ ID NO: 4 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 423 to 846 of SEQ ID NO: 5 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 440 to 865 of SEQ ID NO: 6 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 447 to 864 of SEQ ID NO: 7 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them, and

- amino acid residues 457 to 880 of SEQ ID NO: 8 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them.

It is understood that the Clostridia H _N domain of the invention has the ability to translocate the light chain into the cytoplasm of the target cell.

In one embodiment, the Clostridia L and H _N domains are of the same Clostridia serotype.

In one embodiment, the Clostridia L and H _N domains are from different Clostridia serotypes.

In one embodiment, the hybrid neurotoxin contains a targeting moiety.

As used herein, the term “targeting fragment” (or “targeting domain”) means a fragment that has the ability to bind to a receptor on a target cell. Preferably, the targeting moiety is capable of binding to a protein receptor on the target cell.

Suitable targeting fragments include targeting domains of bacterial toxins, such as Clostridia _HC or _HCC domains, peptides, antibodies, or antibody fragments.

In one embodiment, the hybrid neurotoxin contains a targeting fragment (TM) that binds to a receptor that does not bind Clostridial neurotoxin. TM can replace part or all of the H _C - or H _CC - domain of the heavy chain of the Clostridial neurotoxin. Hybrid neurotoxins containing non-clostridia TM may be referred to as "retargeted neurotoxins" (or "targeted secretion inhibitors", "TSI", "TVEMP" or "TEM"). Examples of TMs suitable for retargeted neurotoxins are disclosed in WO96/33273, WO98/07864, WO00/10598, WO01/21213, WO01/53336, WO02/07759, WO2005/023309, WO2006/026780, WO2006/0952606, WO2006/026780, WO2006/09526090, WO2006/059105, WO2006/059113, WO2007/138339, WO2007/106115, WO2007/106799, WO2009/150469, WO2009/150470, WO2010/055358, WO2010/020811, WO2010/138379, WO2010/138395, WO2010/138382, WO2011/ 020052, Wo2011/020056, Wo2011/020114, Wo2011/020117, Wo2011/11/11/11, 2012/156743, Wo2012/134900, WO2012/134897, Wo2012/134904, Wo2012/134902, Wo2012/135343, Wo2012/13548, Wo2012/135304, WO2012/135304, Wo2012/135304, Wo2012/135304, Wo2012/135304, WO20 WO2012/134902, WO2014/033441, WO2014/128497, WO2014/053651, WO2015/004464, all of which are incorporated herein by reference.

In one embodiment, the hybrid neurotoxin contains the H _CN and/or H _CC domain of Clostridia. Preferably, the Clostridia HCN and/or _HCC domain is _BoNT type A, type B, type C1, type D, type E, type F or type G, or TeNT.

In one embodiment, the hybrid neurotoxin contains a Clostridia _HCN domain that contains a sequence selected from:

- amino acid residues 873 to 1094 of SEQ ID NO: 1 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 860 to 1081 of SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 868 to 1095 of SEQ ID NO: 3 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 864 to 1082 of SEQ ID NO: 4 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 847 to 1069 of SEQ ID NO: 5 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 866 to 1087 of SEQ ID NO: 6 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 865 to 1089 of SEQ ID NO: 7 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them, or

- amino acid residues 881 to 1111 of SEQ ID NO: 8 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them.

In one embodiment, the hybrid neurotoxin contains a Clostridial _HCC domain that contains a sequence selected from:

- amino acid residues 1095 to 1296 of SEQ ID NO: 1 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1082 to 1291 of SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1096 to 1291 of SEQ ID NO: 3 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1083 to 1276 of SEQ ID NO: 4 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1070 to 1252 of SEQ ID NO: 5 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1088 to 1278 of SEQ ID NO: 6 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them,

- amino acid residues 1090 to 1297 of SEQ ID NO: 7 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them, or

- amino acid residues 1112 to 1315 of SEQ ID NO: 8 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them.

It is understood that the H _CC domain of the invention is capable of binding to the clostridia neurotoxin protein receptor.

In one embodiment, the Clostridial _HCN and/or _HCC domain is of the same Clostridial serotype as the light chain.

In one embodiment, the Clostridia _HCN and/or _HCC domain is of a different Clostridial serotype compared to the light chain.

In one embodiment, the hybrid neurotoxin comprises a Clostridia H _CN domain and a Clostridia H _CC domain. Accordingly, H _CN - and/or H _CC -domains of clostridium may belong to the same serotype. Accordingly, H _CN - and/or H _CC -domains of clostridia can belong to different serotypes. In one embodiment, the light chain, H _CN - and H _CC -domain of clostridia belong to the same serotype. In one embodiment, the light chain, the H _CN domain is from one serotype and the H _CC domain is from a different serotype.

In one embodiment, the hybrid neurotoxin comprises a Clostridia H _N domain, a Clostridia H _CN domain, and a Clostridia H _CC domain. Accordingly, H _N -, H _CN - and H _CC -domains of clostridia can belong to the same serotype. Accordingly, H _N -, H _CN - and H _CC -domains of clostridia can belong to different serotypes. In one embodiment, the light chain, H _N -, H _CN - and H _CC -domains of Clostridia belong to the same serotype. In one embodiment, the light chain, the H _N and H _CN domains belong to the same serotype and the H _CC domain belongs to a different serotype. In one embodiment, the light chain and H _N -domain of Clostridia belong to the same serotype, and H _CN - and H _CC -domains belong to a different serotype.

In one embodiment, when the hybrid neurotoxin comprises the Clostridial _HCC domain, its ability to bind to gangliosides is reduced or completely suppressed compared to the native Clostridial _HCC domain. This can be achieved, for example, by introducing mutations in the ganglioside binding motif of the H _CC domain.

The "percent sequence identity" between two or more nucleotide or amino acid sequences is a function of the number of identical nucleotides/amino acids at identical positions shared by the aligned sequences. Thus, % identity can be calculated as the number of identical nucleotides/amino acids at each position in the alignment, divided by the total number of nucleotides/amino acids in the aligned sequence, multiplied by 100. When calculating % sequence identity, the number of breaks and the length of each break can also be taken into account, which must be entered to optimize the alignment of two or more sequences. Comparing sequences and determining percent identity between two or more sequences can be performed using certain mathematical algorithms that are known to those skilled in the art, such as a global alignment mathematical algorithm (such as that described by Needleman and Wunsch, J. Mol. Biol. 48 ( 3), 443-453, 1972).

The light chain, H _N , H _CN and H _CC domains may have their origin in a mosaic neurotoxin. The term "mosaic neurotoxin" as used herein refers to a naturally occurring Clostridial neurotoxin that contains at least one functional domain from another type of Clostridial neurotoxin (e.g., a different serotype of Clostridial neurotoxin), which Clostridial neurotoxin typically does not contain at least at least one functional domain. Examples of mosaic neurotoxins are the natural toxins BoNT/DC and BoNT/CD. BoNT/DC comprises the L chain and the H _N domain of serotype D and the H _C domain of serotype C, while BoNT/CD consists of the L chain and the H _N domain of serotype C and the H _C domain of serotype D.

The light chain, H _N , H _CN , and H _CC domains may be derived from a modified neurotoxin and derivatives thereof, including, but not limited to, the neurotoxins described below. The modified neurotoxin or derivative thereof may contain one or more amino acids that have been modified from the native (unmodified) form of the neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the toxin. For example, a modified Clostridial neurotoxin may have modified amino acid sequences in one or more domains relative to a native (unmodified) Clostridial neurotoxin sequence. Such modifications can change the functional properties of the neurotoxin, such as biological activity or stability.

The modified neurotoxin retains at least one of the functions of the neurotoxin, selected from the ability to bind to a low-affinity or high-affinity neurotoxin receptor on a target cell, translocate the endopeptidase portion of the neurotoxin (light chain) into the cytoplasm of the cell, and cleave the SNARE protein. Preferably, the modified neurotoxin retains at least two of these functions. More preferably, the modified neurotoxin retains these three functions.

The modified neurotoxin may contain one or more modifications in the amino acid sequence of the heavy chain (such as a modified _HC domain), where the modified heavy chain binds to target nerve cells with higher or lower affinity than the native (unmodified) neurotoxin. Such modifications in the HC domain may include modification of residues in the ganglioside binding site in the HC domain or protein binding site ( _SV2 or _{synaptotagmin} ) that alters binding to the ganglioside receptor and/or protein receptor of the target nerve cell. Examples of such modified neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are incorporated herein by reference in their entirety.

The modified neurotoxin may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domain, which may alter or modify the specificity for the SNARE protein of the modified LC. Examples of such modified neurotoxins are described in WO 2010/120766 and US 2011/0318385, both of which are incorporated herein by reference in their entirety.

The modified neurotoxin may contain one or more modifications that increase or decrease the biological activity and/or biological stability of the modified neurotoxin. For example, the modified neurotoxin may contain a leucine or tyrosine motif, where this motif increases or decreases the biological activity and/or biological stability of the modified neurotoxin. Suitable leucine motifs include xDxxxLL, xExxxLL, xExxxIL and xExxxLM (where x is any amino acid). Suitable tyrosine motifs include Y-x-x-Hy (where Hy is a hydrophobic amino acid). Examples of modified neurotoxins containing leucine and tyrosine motifs are described in WO 2002/08268, which is incorporated herein by reference in its entirety.

As used herein, the term "selective ganglioside-binding fragment" means a fragment that binds to one type of ganglioside with higher affinity than to other gangliosides. Binding affinity can be quantified by determining the equilibrium dissociation constant or K _d between the ganglioside and the binding moiety (the lower the K _d , the higher the affinity). Methods for determining ganglioside binding affinity are well known in the art and include, for example, surface plasmon resonance (SPR) as described, for example, in Kuziemko, Geoffrey M. et al. "Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance." Biochemistry 35.20 (1996): 6375-6384, or in MacKenzie, C. Roger, et al. "Quantitative analysis of bacterial toxin affinity and specificity for glycolipid receptors by surface plasmon resonance." Journal of Biological Chemistry 272.9 (1997): 5533-5538. In particular, Kuziemko et al. in 1996 (cited above) found using SPR that cholera toxin preferentially binds to gangliosides in the following order: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1, and that the measured affinity The binding (K _d ) of cholera toxin to the ganglioside sequence ranges from 4.61×10 ^-12 M for GM1 to 1.88×10 ^-10 M for asialo-GM1. Mackenzie et al. in 1997 (cited above) determined by surface plasmon resonance (SPR) using the liposome capture method that CtxB binds to GM1 and GD1b with an affinity of 7.3 x 10 ^-10 M and 8 x 10 ^-9 M, respectively. that the heat-labile enterotoxin (LT) from E. coli binds to GM1 and GD1b with an affinity of 5.7×10 ^-10 M and 3.0×10 ^-9 M, respectively, and that the tetanus toxin C fragment binds to GD1b and GT1b with affinity respectively 1.5×10 ^-7 M and 1.7×10 ^-7 m.

Ganglioside binding affinity can also be determined using a competitive ELISA assay, as described, for example, in Sinclair, Haydn R., et al. "Sialyloligosaccharides inhibit cholera toxin binding to the GM1 receptor." Carbohydrate research 343.15 (2008): 2589-2594. Another method for determining binding affinity for a ganglioside is based on the use of a radiolabeled ligand (eg, labeled with ¹²⁵ I) as described, for example, in Nishiki, Tei-ichi, et al. "The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a." FEBS letters 378.3 (1996): 253-257. Another way to determine binding affinity for ganglioside is to use isothermal titration calorimetry as described in Turnbull, W. Bruce, et al. "Dissecting the cholera toxin-ganglioside GM1 interaction by isothermal titration calorimetry." Journal of the American Chemical Society 126.4 (2004): 1047-1054.

In one embodiment, the K _d between the selective ganglioside-binding moiety and the ganglioside is less than about 10 ^-9 M, preferably less than about 10 ^-10 M, more preferably less than about 10 ^-1 1 M, more preferably less than about 5×10 ^-12 M.

Suitable ganglioside-binding moieties (GBMs) include bacterial toxin GBMs (other than clostridia H _C or H _CC domains), peptides, proteins or protein fragments, antibodies or antibody fragments.

In one embodiment, GBM is a peptide. Examples of peptides suitable for use as GBMs include Alzheimer's-accumulating amyloid peptide (Aβ) that binds to GM1, Parkinson's disease-associated protein α-synuclein that binds to GM3, and described chimeric peptides such as α-synuclein/Aβ described in Yahi and Fantini 2014, which binds to GM1 and GM3 PloS one 9.8 (2014): e104751).

In one embodiment, the GBM is a protein or protein fragment. Examples of proteins suitable for use as GBMs include growth factor receptors such as the epidermal growth factor receptor (EGFR) which binds to GM3, GM1, GM2, GM4, GD3, GD1a and GT1b and the vascular endothelial growth factor receptor (VEGFR) , which binds to GM3, GD1a and GT1b (Krengel, Ute, and Paula A. Bousquet. "Molecular recognition of gangliosides and their potential for cancer immunotherapies." Frontiers in Immunology, 2014, vol 5, article 325).

In a preferred embodiment, the GBM is derived from a bacterial toxin, for example, the GBM is selected from the B-subunit of cholera toxin (CtxB) and heat-labile enterotoxin (LT) of E. coli .

GM1 (or GM1a) is the only known receptor for the cholera toxin B subunit (CtxB). Therefore, by introducing the B-subunit of cholera toxin into the Clostridial neurotoxin or its fragment, it can be selectively targeted to neurons containing GM1. GM1 is also a heat-labile enterotoxin receptor from Escherichia coli . (Zoeteweij, J. Paul, et al. "GM1 binding-deficient exotoxin is a potent noninflammatory broad spectrum intradermal immunoadjuvant." The Journal of Immunology 177.2 (2006): 1197-1207).

It is also believed that the hybrid neurotoxin of the invention, wherein the GBM is derived from a bacterial toxin, is particularly suitable for local delivery of the hybrid neurotoxin. Indeed, it has also been shown that bacterial exotoxins can be safely applied topically to human skin (Zoeteweij, J. Paul, et al. "GM1 binding-deficient exotoxin is a potent noninflammatory broad spectrum intradermal immunoadjuvant." The Journal of Immunology 177.2 (2006) : 1197-1207).

In one embodiment, the ganglioside is GM1 and the GBM is selected from CtxB and E. coli thermolabile enterotoxin (LT).

Cholera toxin (CT) is secreted by the gram-negative bacterium Vibrio cholerae . CT belongs to a larger family of AB toxins that have an enzymatically active A domain responsible for toxicity induction and a cell binding B domain responsible for cell entry. CT belongs to the AB ₅ subfamily, which consists of six polypeptides, one A-subunit and a homopentameric B-subunit, which self-assemble to form a cholotoxin prior to secretion. Other AB ₅ toxins include heat-labile enterotoxins, Shiga toxin, Shiga-like toxins, and pertussis toxin. The CT subunits, A and B (CtxA and CtxB, respectively), are non-covalently linked. The 27 kDa A subunit contains a serine protease cleavage site located between residues 192 and 195, which allows the A subunit to be cleaved into two polypeptides: the A2 chain and the A1 chain. A disulfide bond between residues 187 and 199 links these chains together. The A1 chain is responsible for the enzymatic activity of CT. Five 11.5 kDa B subunits assemble non-covalently to form a homopentamer that binds to the GM1 ganglioside at the plasma membrane. The B ₅ -subunit-GM1 complex transports the A-subunit to the endoplasmic reticulum. After retrotranslocation, the A1 chain enters the cytosol as an active ADP-ribosyl transferase, which modifies the heterotrimeric G protein Gsα. Modification of this G protein results in constitutive activation of adenylate cyclase and rapid production of cAMP. In the cells of the intestine, this causes the secretion of chloride in the intestine, which is accompanied by a massive movement of water and diarrhea, which is the hallmark of cholera. ((Wernick, Naomi LB, et al. "Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum." Toxins 2.3 (2010): 310-325.)

An exemplary amino acid sequence of the cholera toxin B subunit is shown as SEQ ID NO: 9 (UniProtKB accession number P01556), which consists of a signal peptide (amino acid residues 1 to 21 in SEQ ID NO: 9) and a B subunit (amino acid residues 22 to 124 in SEQ ID NO: 9).

An example of the amino acid sequence of the cholera toxin A subunit is shown as SEQ ID NO: 10 (UniProtKB accession number P01555), which consists of a signal peptide (amino acid residues 1 to 18 in SEQ ID NO: 10), an A1 domain (amino acid residues with 19 to 212 in SEQ ID NO: 10) and the A2 domain (amino acid residues 213 to 258 in SEQ ID NO: 10).

The monomeric B subunit has been shown to be sufficient for cell binding and intoxication (Jobling, Michael G., et al. "A single native ganglioside GM1-binding site is sufficient for cholera toxin to bind to cells and complete the intoxication pathway." MBio 3.6 (2012): e00401-12).

The inventors have shown that the addition of a CtxB domain to an endonegative BoNT/A1 (BoNT/A1(0)) endows BoNT/A1 with the ability to bind to GM1, a ganglioside that is not a natural receptor for BoNT/A1.

Without wishing to be bound by any theory, the authors believe that the CtxB subunit will result in increased potency due to cholera toxin having a greater binding affinity for GM1 than that of BoNT for its respective gangliosides. For example, the binding affinity of BoNT/B to the GT1b/GD1a-associated synaptotagmin complex (dual receptor model) has been shown to be in the nanomolar range (“high affinity 0.4 nM, low affinity 4.1 nM”) (Nishiki et al, FEBS Letters 1996), which is 1000 times higher than the picomolar binding affinity of Ctx-B and GM1 published in Kuzimeko et al., Biochemistry 1996.

The CtxB domain of the invention preferably contains amino acid residues 22 to 124 of SEQ ID NO: 9 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with them. It is understood that the CtxB domain of the invention is capable of binding to GM1.

In a preferred embodiment, the selective ganglioside-binding moiety contains one or more B-subunits of cholera toxin (CtxB).

In one embodiment, the light chain is covalently linked to said one or more cholera toxin B subunits (CtxB).

In one embodiment, the selective ganglioside binding moiety contains one CtxB. In one embodiment, the selective ganglioside binding fragment contains two CtxB. In one embodiment, the selective ganglioside binding moiety contains three CtxBs. In one embodiment, the selective ganglioside binding moiety contains four CtxBs. In one embodiment, the selective ganglioside binding moiety contains five CtxB.

In one embodiment, the selective ganglioside binding moiety contains one or more CtxBs that are located at the C-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety contains one or more CtxBs that are located at the N-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety consists of a single CtxB that is located at the C-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety consists of a single CtxB that is located at the N-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety consists of two CtxBs that are located at the C-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety consists of two CtxBs that are located at the N-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety consists of three CtxBs that are located at the C-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety consists of three CtxBs that are located at the N-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety consists of four CtxBs that are located at the C-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside binding fragment consists of four CtxBs that are located at the N-terminus of the light chain of the Clostridial neurotoxin. In one embodiment, the selective ganglioside binding moiety consists of five CtxBs that are located at the C-terminus of the light chain of a Clostridial neurotoxin. In one embodiment, the selective ganglioside-binding moiety consists of five CtxBs that are located at the N-terminus of the light chain of a Clostridial neurotoxin.

In one embodiment, the hybrid neurotoxin comprises a Clostridial H _N domain and the selective ganglioside binding fragment comprises one or more CtxBs that are located at the C-terminus of the Clostridial H _N domain. In another embodiment, one or more CtxBs are located at the N-terminus of the H _N -domain of Clostridia. In one embodiment, the selective ganglioside-binding moiety consists of a single CtxB that is located at the C-terminus of the H _N -domain of Clostridia. In one embodiment, the selective ganglioside binding moiety consists of a single CtxB that is located at the N-terminus of the Clostridia H _N domain. In one embodiment, the selective ganglioside binding moiety consists of two CtxBs that are located at the C-terminus of the H _N -domain of Clostridia. In one embodiment, the selective ganglioside binding moiety consists of two CtxBs that are located at the _N -terminus of the Clostridia HN-domain. In one embodiment, the selective ganglioside-binding moiety consists of three CtxBs that are located at the C-terminus of the H _N -domain of Clostridia. In one embodiment, the selective ganglioside binding moiety consists of three CtxBs that are located at the N-terminus of the H _N -domain of Clostridia. In one embodiment, the selective ganglioside binding moiety consists of four CtxBs that are located at the C-terminus of the H _N -domain of Clostridia. In one embodiment, the selective ganglioside-binding moiety consists of four CtxBs that are located at the N-terminus of the H _N -domain of Clostridia. In one embodiment, the selective ganglioside-binding moiety consists of five CtxBs that are located at the C-terminus of the H _N -domain of Clostridia. In one embodiment, the selective ganglioside-binding moiety consists of five CtxBs that are located at the N-terminus of the H _N -domain of Clostridia.

In one embodiment, the selective ganglioside binding moiety comprises one or more cholera toxin B subunits (CtxB) and the hybrid neurotoxin further comprises a cholera toxin A2 subunit (CtxA2). Preferably, CtxA2 is covalently linked to the Clostridia light chain. In addition, it is preferred that CtxA2 is covalently linked to the Clostridial neurotoxin light chain and CtxB is non-covalently linked to the Clostridial neurotoxin light chain. In particular, it is thought that the cholera toxin A2 subunit (CtxA2) can act as a linker, forming a non-covalent bond with the B subunit pentamer (CtxB5), which will bind to the ganglioside on the target cell and internalize the Clostridial neurotoxin light chain into the cell.

Without wishing to be bound by any theory, the authors suggest that such embodiments, where CtxB binds to the hybrid neurotoxin through a non-covalent bond (to the CtxA2 subunit), allow for a pentameric arrangement of the CtxB subunit (CtxB ₅ ) and thus cause increased binding affinity hybrid neurotoxin with GM1.

The CtxA2 domain of the invention preferably contains amino acid residues 213 to 258 of SEQ ID NO: 10 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99 % sequence identity to them. It is understood that the CtxA2 domain of the invention is capable of binding to the CtxB domain. Preferably, the CtxA2 domain contains residues 255 to 258 (KDEL) of SEQ ID NO: 10.

The CtxA2 domain can be located at the C-terminus or N-terminus of the Clostridial neurotoxin light chain.

In one embodiment, the selective ganglioside-binding moiety comprises one or more cholera toxin B subunits (CtxB) that are non-covalently linked to the Clostridial neurotoxin light chain, and the hybrid neurotoxin further comprises a cholera toxin A2 subunit (CtxA2) and an H _N -domain that is covalently associated with the light chain of Clostridial neurotoxin. In one embodiment, the CtxA2 domain is located at the N-terminus of the H _N -domain of Clostridia and is preferably located between the activation site and the H _N -domain ("central location"). In one embodiment, the CtxA2 domain is located at the C-terminus of the Clostridia H _{N -domain} , and when the hybrid neurotoxin comprises an _HCN- and/or HCC-domain, the ganglioside-binding moiety may be located at the C-terminus or N-terminus of the _HCN- and /or H _CC -domain. The hybrid neurotoxin may contain a linker between the CtxA2 domain and the L-, _HN- , _HCN- and/or HCC-domain.

In one embodiment, the Clostridial neurotoxin light chain is covalently linked to a selective ganglioside binding moiety. The selective ganglioside binding moiety can be located at the C-terminus or N-terminus of the Clostridia light chain.

In one embodiment, the hybrid neurotoxin contains a Clostridia H _N domain and the Clostridia H _N domain is covalently linked to a selective ganglioside binding moiety. The selective ganglioside binding moiety can be located at the C-terminus or N-terminus of the H _N -domain of Clostridia. When the selective ganglioside-binding moiety is located at the N-terminus of the Clostridia H _N domain, it is preferably located between the activation site and the H _N domain ("central location").

When the selective ganglioside-binding fragment is located at the C-terminus of the Clostridia H _{N -domain} , and when the hybrid neurotoxin further comprises an HCN-domain and/or an _HCC -domain, the ganglioside-binding fragment may be located at the C-terminus or N-terminus of the _HCN- or H _CC -domain.

The hybrid neurotoxin may contain a linker between the ganglioside binding domain and the L-, _HN- , _HCN- and/or HCC-domain. Without wishing to be bound by any theory, the authors suggest that the presence of the linker may increase the stability of the hybrid neurotoxin and/or the availability of the ganglioside-binding moiety for its target ganglioside and/or increase expression.

Examples of suitable linkers include GS linkers of various lengths, for example GS5, GS10, GS15, GS18 and GS20, N10, HX27, (EAAAK) ₃ and A(EAAAK) ₄ ALEA(EAAAK) ₄ A , in Chen, Xiaoying, et al. "Fusion protein linkers: property, design and functionality." Advanced drug delivery reviews 65.10 (2013): 1357-1369, incorporated herein by reference.

Examples of the structural arrangement of the hybrid neurotoxins of the invention are given below (GBM: ganglioside binding fragment; TD: translocation domain; BD: protein receptor binding domain; L, HN, HCN, HCC: clostridium domains described herein; AS: activation site; left to the right: from C-terminus to N-terminus):

L-GBM

GBM-L

L-AS-GBM

GBM-AS-L

L-AS-GBM-TD

L-AS-TD-GBM

GBM-TD-AS-L

TD-GBM-AS-L

GBM-L-AS-TD

L - AS - GBM - TD - BD

L - AS - BD - TD - GBM

GBM-TD-BD-AS-L

BD-TD-GBM-AS-L

L-AS-GBM-HN

L-AS-HN-GBM

GBM-HN-AS-L

HN-GBM-AS-L

GBM-L-AS-HN

L - AS - GBM - HN - HCN

L - AS - GBM - HN - HCN - HCC

L-AS-HN-GBM

L-AS-HN-HCN-GBM

L - AS - HN - HCN - HCC - GBM

L - AS - GBM - linker - HN

L - AS - GBM - linker - HN - HCN

L - AS - GBM - linker - HN - HCN - HCC

L - AS - HN - linker - GBM

L - AS - HN - HCN - linker - GBM

L - AS - HN - HCN - HCC - linker - GBM

GBM-L-AS-HN-HCN-HCC

L - AS - linker - GBM - HN

L - AS - linker - GBM - HN - HCN

L - AS - linker - GBM - HN - HCN - HCC

L - AS - linker - GBM - linker - HN

L - AS - linker - GBM - linker - HN - HCN

L - AS - linker - GBM - linker - HN - HCN - HCC

In one embodiment, the hybrid neurotoxin of the invention comprises an H _N domain and is in double stranded form and contains a disulfide bond between the L domain and the H _N domain.

Preferably, the structural arrangement of the hybrid neurotoxin is such that the GBM has a free N- or C-terminus. In embodiments that include an activation site (AS) that allows conversion of the hybrid neurotoxin to the double-stranded form, the structural arrangement of the hybrid neurotoxin is preferably such that the GBM has a free N- or C-terminus after conversion to the double-stranded form.

In embodiments containing a protein receptor (BD) binding domain, such as an H _C or H _CC domain, the structural arrangement of the hybrid neurotoxin is preferably such that the BD has a free N-terminus or C-terminus, and more preferably that both GBM and BD had a free N- or C-terminus. In embodiments involving an activation site (AS) allowing conversion of the hybrid neurotoxin to the double-stranded form, the structural arrangement of the hybrid neurotoxin is preferably such that the BD has a free N- or C-terminus upon conversion to the double-stranded form, and more preferably both GBMs and BD had a free N-terminus or C-terminus after being converted to a double-stranded form.

Hybrid neurotoxins of the present invention can be obtained using recombinant technologies. Thus, in one embodiment, the hybrid neurotoxin of the invention is a recombinant hybrid neurotoxin.

In another aspect, the invention relates to a nucleotide sequence encoding a hybrid neurotoxin according to the invention, for example, a DNA or RNA sequence. In a preferred embodiment, the nucleotide sequence is a DNA sequence.

Nucleic acid molecules of the invention may be prepared using any suitable method known in the art. Thus, nucleic acid molecules can be obtained using chemical synthesis methods. Alternatively, the nucleic acid molecules of the invention may be prepared using molecular biology techniques.

The DNA sequence of the present invention is preferably constructed in silico and then synthesized by conventional DNA synthesis methods.

The above nucleotide sequence is optionally modified to change codons according to the expression system of the final host cell (eg E. coli ) to be used.

In another aspect, the invention relates to a vector containing the nucleotide sequence of the invention. In one embodiment, the nucleotide sequence is obtained as part of a DNA vector containing a promoter and a terminator. In a preferred embodiment, the vector has a promoter selected from Tac, AraBAD, T7-Lac, or T5-Lac.

The vector may be suitable for in vitro and/ or in vivo expression of the above nucleotide sequence. The vector may be a vector for transient and/or stable gene expression. The vector may further contain regulatory elements and/or selectable markers. The vector may be of viral origin, phage origin, or bacterial origin. For example, the expression vector may be a pET vector, pJ401, pGEX, or a derivative thereof.

In another aspect, the invention relates to a cell containing the nucleotide sequence or vector of the invention. Suitable cell types include prokaryotic cells such as E. coli and eukaryotic cells such as yeast cells, mammalian cells, insect cells. Preferably the cell is E. coli .

The hybrid neurotoxins of the invention are particularly suitable for use in therapy.

syndrome: pathogenesis, diagnosis, treatment and prognosis." Nature reviews. Neurology 10.8 (2014): 469; Willison, Hugh J., and Jaap J. Plomp. "Anti-ganglioside Antibodies and the Presynaptic Motor Nerve Terminal." Annals of the New York Academy of Sciences 1132.1 (2008): 114-123). Также было показано, что GM1 присутствует в околоушных железах (слюнных железах) (Nowroozi, Nakisa, et al. "HIGH LEVELS OF GM 1-GANGLIOSIDE AND GM 1-GANGLIOSIDE β-GALACTOSIDASE IN THE PAROTID GLAND: A New Model for Secretory Mechanisms of the Parotid Gland." Otolaryngologic Clinics of North America 32.5 (1999): 779-791).Guillain-Barré syndrome is an acute inflammatory disease that affects the peripheral nervous system and is caused by the binding of antibodies produced by the immune system to gangliosides. Based on the results of clinical subtypes of Guillain-Barré syndrome, gangliosides GM1a, GM1b, GD1a, GalNAc-GD1a are associated with the neuromuscular junction of the extremities, and gangliosides GT1a, GQ1b are associated with the neuromuscular junction of the head and neck ((Van Den Berg, Bianca, et al."

syndrome: pathogenesis, diagnosis, treatment and prognosis." Nature reviews. Neurology 10.8 (2014): 469; Willison, Hugh J., and Jaap J. Plomp. "Anti-ganglioside Antibodies and the Presynaptic Motor Nerve Terminal." Annals of the New York Academy of Sciences 1132.1 (2008): 114-123) GM1 has also been shown to be present in the parotid (salivary glands) (Nowroozi, Nakisa, et al. "HIGH LEVELS OF GM 1-GANGLIOSIDE AND GM 1-GANGLIOSIDE β-GALACTOSIDASE IN THE PAROTID GLAND: A New Model for Secretory Mechanisms of the Parotid Gland." Otolaryngologic Clinics of North America 32.5 (1999): 779-791).

It has been reported that GM1 and GM2 concentrations in lipid rafts from the frontal and temporal cortex were higher in patients with Alzheimer's disease (AD). GM1 clustering has been demonstrated in dorsal root ganglion neurons (sensory neurons). ((Aureli, Massimo, et al. "GM1 ganglioside: past studies and future potential." Molecular neurobiology 53.3 (2016): 1824-1842.)

The gangliosides NeuAc GM3, NeuGc GM3, GM2, GM1, GD3, and GD2 have been shown to be expressed in human tumor cells (Krengel, Ute, and Paula A. Bousquet. "Molecular recognition of gangliosides and their potential for cancer immunotherapies." Frontiers in Immunology , July 2014, vol. 5, article 325).

In one embodiment, the selective ganglioside binding moiety binds to one or more gangliosides selected from GM1a, GM1b, GD1a, and GalNAc-GD1a. Such embodiments are believed to be particularly suitable for the treatment of limb disorders such as upper limb spasticity, lower limb spasticity, focal limb dystonia, limb sprain, repetitive traumatic sprain (RSI), cumulative injury, or occupational overuse syndrome. Indeed, without wishing to be bound by any theory, the authors suggest that targeting the hybrid neurotoxin to gangliosides present at the neuromuscular limb junction would increase selectivity for the limb neuromuscular junction and avoid side effects due to non-specific effects.

In one embodiment, the selective ganglioside binding moiety binds to one or more gangliosides selected from GT1a and GQ1b. Such embodiments are believed to be particularly suitable for the treatment of head and neck disorders such as cervical dystonia, blepharospasm, migraine, myofascial pain, strabismus, hemifacial spasm, eyelid disease, spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, bruxism, and dysphagia. Indeed, without wishing to be bound by any theory, the authors suggest that targeting the hybrid neurotoxin to gangliosides found in the neuromuscular junctions of the head and neck allows increased selectivity for the neuromuscular junctions of the head and neck and avoids side effects due to nonspecific effects.

In one embodiment, the selective ganglioside binding moiety binds to GM1. It is believed that this option is particularly suitable for the treatment of sialorrhea (excessive salivation, drooling). It is also contemplated that this embodiment may be suitable for the treatment of patients suffering from Alzheimer's disease or other neurological disorders.

In one embodiment, the selective ganglioside binding moiety binds to one or more gangliosides selected from NeuAc GM3, NeuGc GM3, GM2, GM1, GD3, and GD2. Such embodiments are believed to be particularly suitable for the treatment of cancer.

In another aspect, the invention relates to a pharmaceutical composition containing a hybrid neurotoxin according to the invention. Preferably, the pharmaceutical composition contains a hybrid neurotoxin together with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.

In another aspect, the invention relates to a hybrid neurotoxin or pharmaceutical composition of the invention for use in therapy.

The hybrid neurotoxin or pharmaceutical composition of the invention is suitable for use in the treatment of a disease associated with unwanted neuronal activity, for example a disease selected from the group consisting of spastic dysphonia, spastic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal dystonia hand, blepharospasm, strabismus, hemifacial spasm, eyelid disease, cerebral palsy, focal spasticity and other voice disorders, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremor, bruxism, dysphagia, anal fissures, achalasia, dysphagia and others disorders of muscle tone and other disorders characterized by involuntary contractions of muscle groups, lacrimation, hyperhidrosis, excessive salivation, excessive secretion in the gastrointestinal tract, secretory disorders, pain from muscle cramps, headache, migraine and dermatological diseases.

In another aspect, the invention relates to the non-therapeutic use of a hybrid neurotoxin or pharmaceutical composition of the invention for the treatment of an aesthetic or cosmetic disorder. According to this aspect of the invention, the subject to be treated for an aesthetic or cosmetic disorder preferably does not suffer from any of the pathological disorders or conditions as described herein. More preferably, said subject is a healthy subject (ie not suffering from any pathological disease or condition).

In another aspect, the invention provides a hybrid neurotoxin comprising a clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a _{Clostridia HCC} or HC domain, and wherein said selective ganglioside binding moiety binds to one or more gangliosides selected from GM1a, GM1b, GD1a and GalNAc-GD1a, for use in the treatment of limb disorders associated with unwanted neuronal activity. In one embodiment, the limb disease is selected from upper limb spasticity, lower limb spasticity, and focal hand dystonia. In a preferred embodiment, the ganglioside is GM1a.

In another aspect, the invention provides a hybrid neurotoxin comprising a clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a _{Clostridia HCC} or HC domain, and wherein said selective ganglioside binding moiety binds to one or more gangliosides selected from GT1a and GQ1b, for use in the treatment of head or neck disease associated with unwanted neuronal activity. In one embodiment, the head or neck disease is selected from cervical dystonia, blepharospasm, migraine, myofascial pain, strabismus, hemifacial spasm, eyelid disease, spastic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, bruxism, and dysphagia.

In another aspect, the invention provides a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a Clostridia HCC or _HC domain, and wherein said selective ganglioside binding moiety binds to GM1, for use in the treatment of _sialorrhea . (or excessive salivation or drooling).

In another aspect, the invention provides a hybrid neurotoxin comprising a clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a _{Clostridia HCC} or HC domain, and wherein said selective ganglioside binding moiety binds to one or more gangliosides selected from NeuAc GM3, NeuGc GM3, GM2, GM1, GD3 and GD2, for use in the treatment of cancer.

In another aspect, the present invention provides a method of treatment comprising administering a therapeutically effective amount of a hybrid neurotoxin or pharmaceutical composition of the invention to a patient in need thereof.

In another aspect, the present invention relates to a method of treating a limb disease associated with unwanted neuronal activity comprising administering to a patient in need thereof a therapeutically effective amount of a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not _HCC - or H _C -domain of Clostridium, and where the specified selective ganglioside-binding fragment binds to one or more gangliosides selected from GM1a, GM1b, GD1a and GalNAc-GD1a.

In another aspect, the present invention relates to a method of treating a head or neck disorder associated with unwanted neuronal activity, comprising administering to a patient in need thereof a therapeutically effective amount of a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not _HCC - or H _C -domain of Clostridia, and where the specified selective ganglioside-binding fragment binds to one or more gangliosides selected from GT1a and GQ1b.

In another aspect, the present invention relates to a method of treating sialorrhea (or excessive salivation or drooling) comprising administering to a patient in need thereof a therapeutically effective amount of a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not _HCC - or H _C -domain of Clostridia, and where the specified selective ganglioside-binding fragment binds to GM1.

In another aspect, the present invention relates to a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of a hybrid neurotoxin comprising a Clostridium neurotoxin light chain and a selective ganglioside binding moiety, wherein the selective ganglioside binding moiety is not a _{Clostridial HCC} or HC domain, and where the specified selective ganglioside-binding fragment binds to one or more gangliosides selected from NeuAc GM3, NeuGc GM3, GM2, GM1, GD3 and GD2.

It should be understood that all other miscellaneous features relating to the hybrid neurotoxin described herein and the preferred embodiments apply mutatis mutandis to the therapeutic and cosmetic aspects of the invention.

It should also be understood that the pharmaceutical composition according to the invention can be used for therapeutic and cosmetic purposes according to the invention.

The genetically engineered hybrid neurotoxins of the present invention may be presented as oral, parenteral, continuous infusion, inhalation, or topical preparation. Compositions suitable for injection may be presented in the form of solutions, suspensions or emulsions, or dry powders, which are dissolved or resuspended in a suitable vehicle before use.

In the case of a hybrid neurotoxin to be administered locally, the hybrid neurotoxin can be formulated as a cream (eg, for topical use) or formulated for subcutaneous injection.

Means for topical delivery may include an aerosol or other spray (eg, nebulizer). In this respect, the aerosol formulation of the hybrid neurotoxin provides delivery to the lungs and/or other nasal and/or bronchial or airways.

Hybrid neurotoxins according to the invention can be administered to a patient by intrathecal or epidural injection into the spinal column at the level of the spinal segment involved in the innervation of the affected organ.

The preferred route of administration is laparoscopic and/or localized, in particular intramuscular, injection.

Dose ranges for administering the neurotoxins of the present invention are such as to provide the desired therapeutic effect. It is understood that the required dosage range will depend upon the nature of the particular hybrid neurotoxin or composition, the route of administration, the type of formulation, the age of the patient, the nature, extent or severity of the patient's disease, the contraindications, if any, and the judgment of the attending physician. Changes in these dosage levels can be adjusted using standard empirical procedures for optimization.

Liquid dosage forms are usually made using a hybrid neurotoxin and a pyrogen-free sterile carrier. The genetically engineered hybrid neurotoxin, depending on the carrier and concentration used, can be either dissolved or resuspended in the carrier. When making solutions, the hybrid neurotoxin can be dissolved in the vehicle, the solution being made isotonic if necessary by adding sodium chloride and sterilized by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if the solution is sufficiently stable in sealed containers, it can be sterilized in an autoclave. Preferably, additives such as buffering, solubilizing, stabilizing, preservative or bactericidal, suspending or emulsifying agents and/or local anesthetics can be dissolved in the carrier.

Dry powders which are dissolved or resuspended in a suitable vehicle before use can be prepared by filling the pre-sterilized ingredients into a sterile container using aseptic technique in a sterile area. Alternatively, the ingredients may be dissolved in suitable containers using aseptic technique in a sterile area. The product is then freeze-dried and the containers sealed.

Parenteral suspensions suitable for intramuscular, subcutaneous or intradermal injection are made in essentially the same manner, except that the sterile components are resuspended in a sterile vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration. The components may be isolated in a sterile state or, alternatively, they may be sterilized after isolation, for example, by gamma irradiation.

When administered according to the present invention, various delivery technologies can be used, including microparticulate encapsulation, viral delivery systems, or high pressure aerosol administration.

This invention is not limited to the examples of methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of this invention. Numeric ranges include numbers that define the range. Unless otherwise indicated, any nucleotide sequences are written from left to right in the 5'-3' orientation; amino acid sequences are written from left to right in the orientation from the amino-terminus to the carboxyl-terminus, respectively.

When a range of values is given, it is understood that each intermediate value is also specifically disclosed, to the nearest tenth of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range. Every smaller range between any claimed value or intermediate value in the specified range and any other claimed or intermediate value in the specified range is included in this description. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range in which any, none, or both limits are included in the smaller ranges is also included in this specification, subject to any specifically excluded limit in the stated range. Where the specified range includes one or both of the limits, ranges excluding one or both of these included limits are also included in this description.

It should be noted that the singular forms used herein and in the appended claims include reference to the plural unless the context clearly indicates otherwise. Thus, for example, reference to "clostridial neurotoxin" includes a plurality of such candidate agents, and reference to "a given clostridial neurotoxin" includes reference to one or more clostridial neurotoxins and their equivalents known to those skilled in the art, and so on.

Sequence Information

SEQ ID NO: 1 - BoNT/A1 - UniProtKB accession number P10845 ( Clostridium botulinum)

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 2 - BoNT/B1 - UniProtKB accession number P10844 ( Clostridium botulinum)

MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYIN RWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

SEQ ID NO: 3 - BoNT/C1 - UniProtKB accession number P18640 ( Clostridium botulinum)

MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNN APGYNKWFFVTVTNNMMGNMKIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFVPVSE

SEQ ID NO: 4 - BoNT/D - UniProtKB accession number P19321 ( Clostridium botulinum)

MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTIYTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNSGWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTN KWFFVTITNNIMGYMKLYINGELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQILRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSVSDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNYGIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDPGWVE

SEQ ID NO: 5 - BoNT/E - accession number WP_003372387 ( Clostridium botulinum)

MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEIIWTLQDNAGINQKLAFNYGNANGISDYINKWIFVTITNDRL GDSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMNFKNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

SEQ ID NO: 6 - BoNT/F - UniProtKB access number YP_001390123 ( Clostridium botulinum)

MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLNNNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELLIPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERFITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDCIRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMIS ISDYINKWIFVTITNNRLGNSRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETLYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKLIRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQEN

SEQ ID NO: 7 - BoNT/G - UniProtKB accession number WP_039635782 ( Clostridium botulinum)

MPVNIKNFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFNASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLGNASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGHSPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLYGIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNLAGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAYEEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYNTQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGDSLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVKGVIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAFEIGGAAILMEFIPELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTIKERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFINQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDSIPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGSDVIFNDIGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTIISCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKD NISDYINKWFSITITNDRLGNANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVSSLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAINYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQLFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYDNYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

SEQ ID NO: 8 - TeNT - UniProtKB accession number P04958 ( Clostridium tetani)

MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIGITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNL IWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

SEQ ID NO: 9 - B-subunit of cholera toxin ( Vibrio cholera)

MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN

SEQ ID NO: 10 - A-subunit of cholera toxin ( Vibrio cholera)

MVKIIFVFFIFLSSFSYANDDKLYRADSRPPDEIKQSGGLMPRGQSEYFDRGTQMNINLYDHARGTQTGFVRHDDGYVSTSISLRSAHLVGQTILSGHSTYYIYVIATAPNMFNVNDVLGAYSPHPDEQEVSALGGIPYSQIYGWYRVHFGVLDEQLHRNRGYRDRYYSNLDIAPAADGYGLAGFPPEHRAWREEPWIHHAPPGCGNAPRSSMSNTCDEKTQSLGVKFLDEYQSKVKRQIFSGYQSDIDTHNRIKDEL

SEQ ID NO: 11 - BoNT/A1(0)-CtxBCP ( Artificial Sequence)

MGSMEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHQLIYAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANSGGGGSGGGGSGGGGSPRGSALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINT SILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLRKSFHHHHHH

Hereinafter, the invention will be described by way of example only, based on the following figures and examples.

FIGURES

Figure 1: examples of Ctx-BoNT hybrid neurotoxins

Figure 2: Fractions analyzed by SDS-PAGE after HisTrap HP capture column. The target construct elutes in fractions E3-F6 (250-500 mM imidazole).

Figure 3: fractions analyzed by electrophoresis in SDS-PAGE, after the second chromatographic step, anion exchange chromatography. The target protein is eluted in fractions 13-30 (with increasing NaCl concentration).

Figure 4: Fractions analyzed by SDS-PAGE after activation with enterokinase. Analysis shows that the protein is unstable prior to proteolytic activation, although some of the constructs do remain intact. Activation with enterokinase does indeed cleave the construct between the light and heavy chain, and from analysis by SDS-PAGE, it can be assumed that at least part of the product has the predicted composition, i.e. intact light and heavy chains, and attached GS20 and CtxB in the central position.

Figure 5: Western blot analysis after enterokinase activation. 5A - blots stained with anti-tetrahistidine monoclonal antibody and secondary anti-mouse conjugate; 5B - Blots stained with anti-LcA antibody and secondary anti-rabbit conjugate.

Figure 6: Evaluation of free Ctx-B, BoNT/A1(0)-CtxBCP and BoNT/A1(0) in the GM1 competitive binding assay.

EXAMPLES

Example 1 - Expression and purification of BoNT/A1(0)-CtxBCP

A codon optimized construct (for E. coli ) was constructed based on the CtxB primary protein sequence (residues 22 to 103 in SEQ ID NO: 11) and subcloned into endonegative BoNT/A in plasmid pJ401 with a T5 promoter to generate a construct with centrally located CtxB (BoNT/A1(0)-CtxBCP) with enterokinase (EK) activation site, GS20 linker and C-terminal His tag: L _c A(0)-EK-CtxB-GS20-H _c -A-6HT .

The construct was transformed into E. coli strain BL21 (DE3) in mTB medium (tryptone 12 g/l, yeast extract 24 g/l, dipotassium phosphate 9.4 g/l, monopotassium phosphate 2.2 g/l, Melford) with the addition of glycerol (0 .4%, Sigma), glucosamine (0.2%, Sigma) and 30 μg/ml kanamycin (Sigma). A single colony was selected to infect a vial with a microbeaded microbank. Infected beads were stored at -80°C until use. One bead was used to infect 100 ml mTB medium at 37°C. When the optical density at 600 nm reached 4.6, 10 ml of 100 ml culture was added to 1 liter of medium in a 2 liter flask, and the culture was grown at 37°C to OD 600≥1.0. The temperature was lowered to 16°C and the culture was allowed to cool for 1 hour before adding IPTG to a final concentration of 1 mm. The induction lasted 20 hours. The culture was then collected by centrifugation at 6000×g for 20 minutes at 4°C. The spent carrier was decanted and the precipitate was frozen and stored at -80° C. until required.

The cell pellet was resuspended in lysis buffer to a concentration of 6 ml/g (50 mM Tris, pH 8.0, 200 mM NaCl). Cells were lysed by passing through a homogenizer at 20 kpsi once. Cellular debris and insoluble material were separated by centrifugation at 30,000×g for 30 minutes. The supernatant was collected and applied to a 5 ml HisTrap column (pre-packed with Ni ²⁺ resin and equilibrated with lysis buffer). After loading, the column was washed with 50 ml of lysis buffer before elution of the protein with a stepwise gradient of imidazole concentration: 25 ml of 40 mM imidazole, 50 ml of 80 mM imidazole, 25 ml of 125 mM imidazole, 25 ml of 250 mM imidazole and 25 ml of 500 mM imidazole. Fractions of 2.5 ml were collected throughout the elution, and the location of the target chimera was determined by electrophoresis in SDS-PAGE (figure 2).

Fractions E3-F6 (250-500 mM imidazole) containing the target protein were pooled and desalted using a 53 ml 26/10 desalting column. The material was desalted in QHP binding buffer (50 mM Tris, pH 8.0). The material in the new buffer was stored as a single pool and further purified on an anion exchanger.

For additional purification of the chimera, a 5 ml HiTrap QHP column was used. The column was pre-equilibrated in binding buffer (50 mM Tris, pH 8.0) before applying the pool after desalting. The column was washed for 25 ml with binding buffer, after which the protein was eluted in a linear gradient from 0 to 350 mM NaCl per 100 ml. The column was then washed with 25 ml of buffer with a linear gradient of high salt content of 350 mm - 1 M NaCl. Fractions of 2.5 ml were collected throughout the elution and analyzed by SDS-PAGE to determine which fractions contained the target protein (figure 3).

Fractions 13-30 containing the target protein were pooled and concentrated before activation with enterokinase for 18 hours at 4°C and the reaction was stopped by adding AEBSF.

This final material was analyzed by electrophoresis in SDS-PAGE (figure 4).

Analysis shows that the protein was unstable prior to proteolytic activation, although some of the constructs do remain intact. Activation with enterokinase does indeed cleave the construct between the light and heavy chain, and it can be assumed from SDS-PAGE analysis that at least a portion of the product has the predicted composition, i.e. intact light and heavy chains, and attached GS20 and CtxB in the central position.

Samples were also analyzed by Western blot to confirm the presence of the light chain and histidine tag (figure 5). The protein was transferred from the gel to a nitrocellulose membrane using a Bio-Rad Trans-Blot Turbo transfer system. Blots were blocked in PBST buffer containing 0.5% BSA. Blots were stained with either monoclonal anti-tetraHis antibody and secondary anti-mouse conjugate, or their anti-LcA antibody and secondary anti-rabbit conjugate. The Super signal substrate was used to generate the signal and the signal was detected on the Pxi 4 gel documentation system. Western blotting gave a positive signal for the full length target product as well as truncated products.

Example 2 Binding of BoNT/A1(0)-CtxBCP to GM1

Briefly, a transparent F96 Maxisorp plate was coated with 100 ng/ml GM1 overnight, blocked with 2% BSA-PBS, and first incubated with free cholera toxin B subunit (free Ctx-B), BoNT/A1(0)-CtxBCP or BoNT/A1(0) at the indicated concentrations. The plates were then incubated with 40 μg/ml cholera toxin B-subunit conjugated with horseradish peroxidase (Ctx-B-HRP). The HRP activity on the plate after washing was determined with a developing solution, and the optical density at 450 nm was determined after stopping the reaction. Data are mean ± standard error of the mean for three wells (Figure 6).

Figure 6 shows that:

BoNT/A1(0) did not compete for Ctx-B (Ctx-B-HRP) binding to GM1 as expected;

Free Ctx-B competed with Ctx-B-HRP as expected with pEC ₅₀ 0.2 μg/ml;

BoNT/A1(0)-CtxBCP was able to compete with Ctx-B-HRP, showing a pEC ₅₀ approximately 100 times lower than free Ctx-B (49 μg/ml).

It can be concluded that the addition of the Ctx-B domain endows BoNT/A1(0) with the ability to bind to GM1, a ganglioside that is not a natural receptor for BoNT/A1(0).

--->

SEQUENCE LIST

<110> IPSEN BIOPHARM LIMITED

<120> HYBRID NEUROTOXINS

<130> IBL 005-EP-PCT

<150> EP16191468.4

<151> 2016-09-29

<160> 11

<170>PatentIn version 3.5

<210> 1

<211> 1296

<212> Protein

<213> Clostridium botulinum

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

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

1100 1105 1110

Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn 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 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> 2

<211> 1291

<212> Protein

<213> Clostridium botulinum

<400> 2

Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn

1 5 10 15

Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg

20 25 30

Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu

35 40 45

Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly

50 55 60

Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn

65 70 75 80

Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe

85 90 95

Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile

100 105 110

Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu

115 120 125

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

130 135 140

Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile

145 150 155 160

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

165 170 175

Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln

180 185 190

Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu

195 200 205

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

210 215 220

Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr

225 230 235 240

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

245 250 255

Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe

260 265 270

Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile

275 280 285

Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn

290 295 300

Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr

305 310 315 320

Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly

325 330 335

Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu

340 345 350

Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys

355 360 365

Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys

370 375 380

Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile

385 390 395 400

Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile

405 410 415

Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr

420 425 430

Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr

500 505 510

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

515 520 525

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

530 535 540

Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp

545 550 555 560

Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp

565 570 575

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

580 585 590

Trp Val Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser

595 600 605

Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys

675 680 685

Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp

690 695 700

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

705 710 715 720

Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr

725 730 735

Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp

740 745 750

Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile

755 760 765

Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met

770 775 780

Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn

785 790 795 800

Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr

805 810 815

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

820 825 830

Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile

835 840 845

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

850 855 860

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

865 870 875 880

Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys

885 890 895

Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr

900 905 910

Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val

915 920 925

Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn

930 935 940

Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser

945 950 955 960

Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile

965 970 975

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

980 985 990

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

995 1000 1005

Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu

1010 1015 1020

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

1025 1030 1035

Glu Ile Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe

1040 1045 1050

Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln

1055 1060 1065

Ser Asn Ile Glu Glu Arg Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr

1070 1075 1080

Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr

1085 1090 1095

Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu Lys

1100 1105 1110

Lys Asp Ser Pro Val Gly Glu Ile Leu Thr Arg Ser Lys Tyr Asn

1115 1120 1125

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

1130 1135 1140

Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp

1145 1150 1155

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

1160 1165 1170

Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys Lys

1175 1180 1185

Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu

1190 1195 1200

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

1205 1210 1215

Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp

1220 1225 1230

Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile

1235 1240 1245

Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr

1250 1255 1260

Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys

1265 1270 1275

Asn Trp Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu

1280 1285 1290

<210> 3

<211> 1291

<212> Protein

<213> Clostridium botulinum

<400> 3

Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn

1 5 10 15

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

20 25 30

Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp

35 40 45

Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val

50 55 60

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

65 70 75 80

Ser Asp Lys Asp Pro Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg

85 90 95

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

100 105 110

Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp

115 120 125

Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn

130 135 140

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

145 150 155 160

Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr

165 170 175

Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile

180 185 190

Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp

195 200 205

Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile

210 215 220

Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly

225 230 235 240

Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile

245 250 255

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

260 265 270

Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr

275 280 285

Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu

290 295 300

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

305 310 315 320

Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser

325 330 335

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

340 345 350

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

355 360 365

Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr

370 375 380

Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn

385 390 395 400

Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser

405 410 415

Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu

420 425 430

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

435 440 445

Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro

450 455 460

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

465 470 475 480

Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser

485 490 495

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

500 505 510

Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly

515 520 525

Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp

595 600 605

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

610 615 620

Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn

625 630 635 640

Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val

645 650 655

Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly

660 665 670

Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys

675 680 685

Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser

690 695 700

Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe

705 710 715 720

Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile

785 790 795 800

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

805 810 815

Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu

820 825 830

Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile

835 840 845

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

850 855 860

Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys

865 870 875 880

Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu

885 890 895

Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly

900 905 910

Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn

915 920 925

Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile

930 935 940

Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp

945 950 955 960

Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe

965 970 975

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

980 985 990

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

995 1000 1005

Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met

1055 1060 1065

Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys

1070 1075 1080

Asp Ile Asn Ile Leu Phe Asn Ser Leu Gln Tyr Thr Asn Val Val

1085 1090 1095

Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr

1100 1105 1110

Met Val Asn Ile Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser

1115 1120 1125

Arg Gln Ile Val Phe Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn

1130 1135 1140

Glu Gly Tyr Lys Ile Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn

1145 1150 1155

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

1160 1165 1170

Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys Asn Glu Thr Met

1175 1180 1185

Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala Ile Gly Leu

1190 1195 1200

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

1205 1210 1215

Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe Lys

1220 1225 1230

Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly

1235 1240 1245

Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr

1250 1255 1260

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

1265 1270 1275

Ser Thr Ser Thr His Trp Gly Phe Val Pro Val Ser Glu

1280 1285 1290

<210> 4

<211> 1276

<212> Protein

<213> Clostridium botulinum

<400> 4

Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp

1 5 10 15

Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr

20 25 30

Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu

35 40 45

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

50 55 60

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

65 70 75 80

Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg

85 90 95

Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val

100 105 110

Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp

115 120 125

Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly

130 135 140

Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly

145 150 155 160

Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly

165 170 175

Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu

180 185 190

Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn

195 200 205

Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val

210 215 220

Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly

225 230 235 240

Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly

245 250 255

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

260 265 270

Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln

275 280 285

Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu

290 295 300

Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp

305 310 315 320

Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn

325 330 335

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

340 345 350

Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu

420 425 430

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

435 440 445

Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys

450 455 460

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

465 470 475 480

Thr Asn Val Gln Asn Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile

485 490 495

Leu Asp Gly Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu

500 505 510

Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val

515 520 525

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

530 535 540

Tyr Leu Glu Ser Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu

545 550 555 560

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

565 570 575

Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly

580 585 590

Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn

595 600 605

Ile Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile

610 615 620

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

625 630 635 640

Gly Asn Phe Asn Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu

645 650 655

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

660 665 670

Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn

675 680 685

Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met

690 695 700

Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe Asn His Ile Asn

705 710 715 720

Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala

725 730 735

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

740 745 750

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

755 760 765

Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val

770 775 780

Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn

785 790 795 800

Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser

805 810 815

His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val

820 825 830

Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr

835 840 845

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

850 855 860

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

865 870 875 880

Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln

885 890 895

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

900 905 910

Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr

915 920 925

Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr

930 935 940

Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser

945 950 955 960

Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln

965 970 975

Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser

980 985 990

Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile Thr

995 1000 1005

Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu

1010 1015 1020

Lys Gln Ser Gln Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp

1025 1030 1035

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

1040 1045 1050

Met Leu Trp Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser

1055 1060 1065

Asn Glu Asp Ile Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn

1070 1075 1080

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

1085 1090 1095

Tyr Tyr Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

Asn Pro Tyr Ser Arg Ile Leu Asn Gly Asp Asn Ile Ile Leu His

1145 1150 1155

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

1160 1165 1170

Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser Gln Asn Cys Val

1175 1180 1185

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

1190 1195 1200

Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr Cys Ser

1205 1210 1215

Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala Asp

1220 1225 1230

Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro

1235 1240 1245

Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser

1250 1255 1260

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

1265 1270 1275

<210> 5

<211> 1252

<212> Protein

<213> Clostridium botulinum

<400> 5

Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg

1 5 10 15

Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser

20 25 30

Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile

35 40 45

Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp

115 120 125

Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu

130 135 140

Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr

145 150 155 160

Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His

165 170 175

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

180 185 190

Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu

195 200 205

Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala

210 215 220

Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu

225 230 235 240

Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly

245 250 255

Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr

260 265 270

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

275 280 285

Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu

290 295 300

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

305 310 315 320

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

325 330 335

Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile

340 345 350

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

355 360 365

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

370 375 380

Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr

385 390 395 400

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

405 410 415

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

420 425 430

Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile

435 440 445

Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr

450 455 460

Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala

465 470 475 480

Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala

485 490 495

Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His

500 505 510

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

515 520 525

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

530 535 540

Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile

545 550 555 560

Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile

565 570 575

Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr

580 585 590

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

595 600 605

Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala

610 615 620

Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu

625 630 635 640

Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser

645 650 655

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

660 665 670

Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn

675 680 685

Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met

690 695 700

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

705 710 715 720

Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Glu Lys Asn Glu Leu Thr Asn

725 730 735

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

740 745 750

Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser

755 760 765

Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu

770 775 780

Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His

785 790 795 800

Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr

805 810 815

Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp

820 825 830

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

835 840 845

Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp

850 855 860

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

865 870 875 880

Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser

885 890 895

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

900 905 910

Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn

915 920 925

Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg

930 935 940

Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile

945 950 955 960

Trp Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr

1025 1030 1035

Thr Arg Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu

1040 1045 1050

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

1055 1060 1065

Thr Asn Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp

1070 1075 1080

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

1085 1090 1095

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

1100 1105 1110

Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser Gly Ile Lys Val Lys

1115 1120 1125

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

1130 1135 1140

Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala Ser Lys Thr His

1145 1150 1155

Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys Glu Lys

1160 1165 1170

Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn Gln Val Val

1175 1180 1185

Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn Phe Lys Asn

1190 1195 1200

Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr

1205 1210 1215

Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr

1220 1225 1230

Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly

1235 1240 1245

Trp Gln Glu Lys

1250

<210> 6

<211> 1278

<212> Protein

<213> Clostridium botulinum

<400> 6

Met Pro Val Val Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp

1 5 10 15

Asp Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys

20 25 30

Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu

35 40 45

Arg Asn Thr Ile Gly Thr Asp Pro Ser Asp Phe Asp Pro Pro Ala Ser

50 55 60

Leu Glu Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr

65 70 75 80

Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys

85 90 95

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

100 105 110

Tyr Ala Lys Pro Tyr Leu Gly Asn Glu His Thr Pro Ile Asn Glu Phe

115 120 125

His Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Ser Ser Thr Asn

130 135 140

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

145 150 155 160

Asp Ile Phe Glu Asn Ser Ser Tyr Pro Val Arg Lys Leu Met Asp Ser

165 170 175

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

180 185 190

Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly

195 200 205

Gly Tyr Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser

210 215 220

Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg

225 230 235 240

Gly Val Thr Tyr Lys Glu Thr Ile Lys Val Lys Gln Ala Pro Leu Met

245 250 255

Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly

260 265 270

Gln Asp Leu Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn

275 280 285

Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Arg Val

290 295 300

Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe

305 310 315 320

Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val

325 330 335

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

340 345 350

Glu Ile Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Asn Asn Arg Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu

450 455 460

Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn

465 470 475 480

Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser

485 490 495

Glu Thr Ile Pro Gln Ile Ser Asn Gln Thr Leu Asn Thr Leu Val Gln

500 505 510

Asp Asp Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile

515 520 525

Glu Glu His Asn Val Val Asp Leu Asn Val Phe Phe Tyr Leu His Ala

530 535 540

Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile

545 550 555 560

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

565 570 575

Glu Phe Ile Asn Thr Ile Asn Lys Pro Val His Ala Ala Leu Phe Ile

580 585 590

Ser Trp Ile Asn Gln Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln

595 600 605

Lys Ser Thr Phe Asp Lys Ile Ala Asp Ile Ser Leu Val Val Pro Tyr

610 615 620

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

625 630 635 640

Lys Glu Ala Phe Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Val

645 650 655

Pro Glu Leu Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe

660 665 670

Ile Gly Ser Ser Glu Asn Lys Asn Lys Ile Ile Lys Ala Ile Asn Asn

675 680 685

Ser Leu Met Glu Arg Glu Thr Lys Trp Lys Glu Ile Tyr Ser Trp Ile

690 695 700

Val Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg Lys

705 710 715 720

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

725 730 735

Val Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Arg Asn Arg

740 745 750

Leu Glu Ser Glu Tyr Asn Ile Asn Asn Ile Arg Glu Glu Leu Asn Lys

755 760 765

Lys Val Ser Leu Ala Met Glu Asn Ile Glu Arg Phe Ile Thr Glu Ser

770 775 780

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

785 790 795 800

Leu Arg Glu Tyr Asp Glu Gly Val Lys Glu Tyr Leu Leu Asp Tyr Ile

805 810 815

Ser Glu His Arg Ser Ile Leu Gly Asn Ser Val Gln Glu Leu Asn Asp

820 825 830

Leu Val Thr Ser Thr Leu Asn Asn Ser Ile Pro Phe Glu Leu Ser Ser

835 840 845

Tyr Thr Asn Asp Lys Ile Leu Ile Leu Tyr Phe Asn Lys Leu Tyr Lys

850 855 860

Lys Ile Lys Asp Asn Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys

865 870 875 880

Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asp

885 890 895

Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Ser Ser

900 905 910

Lys Pro Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn

915 920 925

Gly Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys

930 935 940

Tyr Phe Asn Lys Val Asn Leu Asn Asn Glu Tyr Thr Ile Ile Asp Cys

945 950 955 960

Ile Arg Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Asn Tyr Asn Lys

965 970 975

Ile Ile Trp Thr Leu Gln Asp Thr Ala Gly Asn Asn Gln Lys Leu Val

980 985 990

Phe Asn Tyr Thr Gln Met Ile Ser Ile Ser Asp Tyr Ile Asn Lys Trp

995 1000 1005

Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg Ile

1010 1015 1020

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

1025 1030 1035

Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly

1040 1045 1050

Cys Asn Asp Thr Arg Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe

1055 1060 1065

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

1070 1075 1080

Glu Pro Asp Pro Ser Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu

1085 1090 1095

Leu Tyr Asn Lys Arg Tyr Tyr Leu Leu Asn Leu Leu Arg Thr Asp

1100 1105 1110

Lys Ser Ile Thr Gln Asn Ser Asn Phe Leu Asn Ile Asn Gln Gln

1115 1120 1125

Arg Gly Val Tyr Gln Lys Pro Asn Ile Phe Ser Asn Thr Arg Leu

1130 1135 1140

Tyr Thr Gly Val Glu Val Ile Ile Arg Lys Asn Gly Ser Thr Asp

1145 1150 1155

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

1160 1165 1170

Ile Asn Val Val Asp Arg Asp Val Glu Tyr Arg Leu Tyr Ala Asp

1175 1180 1185

Ile Ser Ile Ala Lys Pro Glu Lys Ile Ile Lys Leu Ile Arg Thr

1190 1195 1200

Ser Asn Ser Asn Asn Ser Leu Gly Gln Ile Ile Val Met Asp Ser

1205 1210 1215

Ile Gly Asn Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly Gly

1220 1225 1230

Asn Ile Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser

1235 1240 1245

Ser Trp Tyr Tyr Asn Asn Ile Arg Lys Asn Thr Ser Ser Ser Asn Gly

1250 1255 1260

Cys Phe Trp Ser Phe Ile Ser Lys Glu His Gly Trp Gln Glu Asn

1265 1270 1275

<210> 7

<211> 1297

<212> Protein

<213> Clostridium botulinum

<400> 7

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

1 5 10 15

Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr

20 25 30

Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile

100 105 110

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

115 120 125

Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln

130 135 140

Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile

145 150 155 160

Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile

165 170 175

Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met

180 185 190

Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu

195 200 205

Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro

210 215 220

Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr

225 230 235 240

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

245 250 255

Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe

260 265 270

Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile

275 280 285

Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn

290 295 300

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

305 310 315 320

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

325 330 335

Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met

340 345 350

Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr

355 360 365

Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys

370 375 380

Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala

385 390 395 400

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

405 410 415

Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg

420 425 430

Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu

435 440 445

Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys

450 455 460

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

465 470 475 480

Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile

485 490 495

Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr

500 505 510

Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys

515 520 525

Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu

530 535 540

Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu

545 550 555 560

Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr

565 570 575

Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala

580 585 590

Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser

595 600 605

Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser Asp Val Ser Ile

610 615 620

Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala

625 630 635 640

Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu

645 650 655

Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr

660 665 670

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

675 680 685

Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu

690 695 700

Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile

705 710 715 720

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

725 730 735

Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met

740 745 750

Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser

755 760 765

Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser

770 775 780

Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu

865 870 875 880

Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val

885 890 895

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

900 905 910

Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe Val Val Tyr Asp Ser

915 920 925

Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr

930 935 940

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

945 950 955 960

Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn

965 970 975

Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile

980 985 990

Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys

995 1000 1005

Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn

1010 1015 1020

Ile Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu Asn

1025 1030 1035

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

1040 1045 1050

Asn Cys Thr Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn

1055 1060 1065

Ile Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser Ser Ser Leu Tyr

1070 1075 1080

Trp Ile Gln Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn

1085 1090 1095

Pro Leu Arg Tyr Asp Thr Gln Tyr Tyr Leu Phe Asn Gln Gly Met

1100 1105 1110

Gln Asn Ile Tyr Ile Lys Tyr Phe Ser Lys Ala Ser Met Gly Glu

1115 1120 1125

Thr Ala Pro Arg Thr Asn Phe Asn Asn Ala Ala Ile Asn Tyr Gln

1130 1135 1140

Asn Leu Tyr Leu Gly Leu Arg Phe Ile Ile Lys Lys Ala Ser Asn

1145 1150 1155

Ser Arg Asn Ile Asn Asn Asp Asn Ile Val Arg Glu Gly Asp Tyr

1160 1165 1170

Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu Ser Tyr Arg Val

1175 1180 1185

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

1190 1195 1200

Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu Gln Ile

1205 1210 1215

Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu Cys

1220 1225 1230

Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe

1235 1240 1245

Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe

1250 1255 1260

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

1265 1270 1275

Lys Leu Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Glu

1280 1285 1290

Gly Trp Thr Glu

1295

<210> 8

<211> 1315

<212> Protein

<213> Clostridium tetani

<400> 8

Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn

1 5 10 15

Asp Thr Ile Ile Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile

20 25 30

Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu

35 40 45

Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser

50 55 60

Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr

65 70 75 80

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

85 90 95

Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile

100 105 110

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

115 120 125

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

130 135 140

Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe

145 150 155 160

Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu

165 170 175

Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser

180 185 190

Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn

195 200 205

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

210 215 220

Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His

225 230 235 240

Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys

245 250 255

Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu

260 265 270

Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys

275 280 285

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

290 295 300

Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp

305 310 315 320

Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser

325 330 335

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

340 345 350

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

355 360 365

Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys

370 375 380

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

385 390 395 400

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

405 410 415

Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val

420 425 430

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

435 440 445

Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly

450 455 460

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

465 470 475 480

Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr

485 490 495

Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile

500 505 510

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

515 520 525

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

530 535 540

Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile

545 550 555 560

Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile

565 570 575

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

580 585 590

Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln

595 600 605

Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr

610 615 620

Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser

625 630 635 640

Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly

645 650 655

Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu

660 665 670

Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu

675 680 685

Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile

690 695 700

Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys

705 710 715 720

Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys

725 730 735

Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile

740 745 750

Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys

755 760 765

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

770 775 780

Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser

785 790 795 800

Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln

805 810 815

Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile

820 825 830

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

835 840 845

Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser

850 855 860

Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile

865 870 875 880

Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile

885 890 895

Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala

900 905 910

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

915 920 925

Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn

930 935 940

Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys

945 950 955 960

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

965 970 975

Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser

980 985 990

Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala

995 1000 1005

Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe

1010 1015 1020

Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn

1025 1030 1035

Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met

1040 1045 1050

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

1055 1060 1065

Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr

1070 1075 1080

Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro

1085 1090 1095

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

1100 1105 1110

Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr

1115 1120 1125

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

1130 1135 1140

Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr

1145 1150 1155

Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu

1160 1165 1170

Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser

1175 1180 1185

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

1190 1195 1200

Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe

1205 1210 1215

Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly

1220 1225 1230

Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu

1235 1240 1245

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

1250 1255 1260

Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp

1265 1270 1275

Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His

1280 1285 1290

Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr

1295 1300 1305

Asp Glu Gly Trp Thr Asn Asp

1310 1315

<210> 9

<211> 124

<212> Protein

<213> Vibrio cholerae

<400> 9

Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser

1 5 10 15

Ala Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu

20 25 30

Tyr His Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr

35 40 45

Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys

50 55 60

Asn Gly Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp

65 70 75 80

Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala

85 90 95

Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys

100 105 110

Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn

115 120

<210> 10

<211> 258

<212> Protein

<213> Vibrio cholerae

<400> 10

Met Val Lys Ile Ile Phe Val Phe Phe Ile Phe Leu Ser Ser Ser Phe Ser

1 5 10 15

Tyr Ala Asn Asp Asp Lys Leu Tyr Arg Ala Asp Ser Arg Pro Pro Asp

20 25 30

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

35 40 45

Phe Asp Arg Gly Thr Gln Met Asn Ile Asn Leu Tyr Asp His Ala Arg

50 55 60

Gly Thr Gln Thr Gly Phe Val Arg His Asp Asp Gly Tyr Val Ser Thr

65 70 75 80

Ser Ile Ser Leu Arg Ser Ala His Leu Val Gly Gln Thr Ile Leu Ser

85 90 95

Gly His Ser Thr Tyr Tyr Ile Tyr Val Ile Ala Thr Ala Pro Asn Met

100 105 110

Phe Asn Val Asn Asp Val Leu Gly Ala Tyr Ser Pro His Pro Asp Glu

115 120 125

Gln Glu Val Ser Ala Leu Gly Gly Ile Pro Tyr Ser Gln Ile Tyr Gly

130 135 140

Trp Tyr Arg Val His Phe Gly Val Leu Asp Glu Gln Leu His Arg Asn

145 150 155 160

Arg Gly Tyr Arg Asp Arg Tyr Tyr Ser Asn Leu Asp Ile Ala Pro Ala

165 170 175

Ala Asp Gly Tyr Gly Leu Ala Gly Phe Pro Pro Glu His Arg Ala Trp

180 185 190

Arg Glu Glu Pro Trp Ile His His Ala Pro Pro Gly Cys Gly Asn Ala

195 200 205

Pro Arg Ser Ser Met Ser Asn Thr Cys Asp Glu Lys Thr Gln Ser Leu

210 215 220

Gly Val Lys Phe Leu Asp Glu Tyr Gln Ser Lys Val Lys Arg Gln Ile

225 230 235 240

Phe Ser Gly Tyr Gln Ser Asp Ile Asp Thr His Asn Arg Ile Lys Asp

245 250 255

Glu Leu

<210> 11

<211> 1430

<212> Protein

<213> Artificial sequence

<220>

<223> BoNT/A1(0)-CtxBCP

<400> 11

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

1 5 10 15

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

20 25 30

Met Gln Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Leu Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr

100 105 110

Ser Ile Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr

115 120 125

Glu Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp

130 135 140

Gly Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser

145 150 155 160

Ala Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu

165 170 175

Asn Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser

180 185 190

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

195 200 205

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

210 215 220

Ala His Gln Leu Ile Tyr Ala Gly His Arg Leu Tyr Gly Ile Ala Ile

225 230 235 240

Asn Pro Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met

245 250 255

Ser Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Thr Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr

355 360 365

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

370 375 380

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

385 390 395 400

Ala Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe

405 410 415

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

420 425 430

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

435 440 445

Lys Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr

450 455 460

Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu

465 470 475 480

Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr

485 490 495

Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys

500 505 510

Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu

515 520 525

Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala

530 535 540

Ile Ala Ala Ile Ser Met Ala Asn Ser Gly Gly Gly Gly Ser Gly Gly

545 550 555 560

Gly Gly Ser Gly Gly Gly Gly Ser Pro Arg Gly Ser Ala Leu Asn Leu

565 570 575

Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Lys Ser

675 680 685

Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser

690 695 700

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

705 710 715 720

Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr

725 730 735

Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala

740 745 750

Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly

755 760 765

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

770 775 780

Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile Pro Val Leu

785 790 795 800

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

805 810 815

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

820 825 830

Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val Asn Thr Gln

835 840 845

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

850 855 860

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

865 870 875 880

Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu Ser Ser Lys

885 890 895

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

900 905 910

Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile Pro Tyr Gly

915 920 925

Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp Ala Leu Leu

930 935 940

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

945 950 955 960

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

965 970 975

Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr Phe Thr Glu

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

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

1100 1105 1110

Thr Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

Ser Asn Asn Ile Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275

Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn

1280 1285 1290

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

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

Gly Asn Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln

1340 1345 1350

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

1355 1360 1365

Asn Asp Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala

1370 1375 1380

Lys Leu Val Ala Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser

1385 1390 1395

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

1400 1405 1410

Gly Trp Gly Glu Arg Pro Leu Arg Lys Ser Phe His His His His

1415 1420 1425

His His

1430

<---

Claims (26)

1. Hybrid polypeptide for binding to ganglioside, containing: a Clostridial neurotoxin light chain (L) comprising an amino acid sequence having at least 95% sequence identity with amino acid residues 1-448 of SEQ ID NO: 1; H _N -domain of Clostridium neurotoxin containing an amino acid sequence having at least 95% sequence identity with amino acid residues 449-872 SEQ ID NO: 1, H _CN is a Clostridial neurotoxin domain containing an amino acid sequence having at least 95% sequence identity with amino acid residues 873-1094 SEQ ID NO: 1; H _CC is a Clostridial neurotoxin domain containing an amino acid sequence having at least 95% sequence identity with amino acid residues 1095-1296 of SEQ ID NO: 1; and a selective ganglioside binding fragment, wherein said selective ganglioside binding fragment is capable of binding to GM1, and wherein the selective ganglioside binding fragment comprises an amino acid sequence having at least 95% sequence identity with amino acid residues 22-124 of SEQ ID NO: 9. 2. A hybrid polypeptide according to claim 1, additionally containing an activation site. 3. The hybrid polypeptide according to claim 2, where the hybrid polypeptide contains from the N-terminus to the C-terminus: a Clostridial neurotoxin light chain (L) comprising an amino acid sequence having at least 95% sequence identity with amino acid residues 1-448 of SEQ ID NO: 1; activation site; a selective ganglioside-binding fragment, wherein said selective ganglioside-binding fragment is capable of binding to GM1 and contains an amino acid sequence having at least 95% sequence identity with amino acid residues 22-124 of SEQ ID NO: 9; H _N -domain of Clostridia neurotoxin containing an amino acid sequence having at least 95% sequence identity with amino acid residues 449-872 SEQ ID NO: 1; H _CN is a Clostridial neurotoxin domain containing an amino acid sequence having at least 95% sequence identity with amino acid residues 873-1094 SEQ ID NO: 1; and H _CC is a Clostridial neurotoxin domain containing an amino acid sequence having at least 95% sequence identity with amino acid residues 1095-1296 of SEQ ID NO: 1. 4. The hybrid polypeptide according to claim 3, further comprising a linker between the selective ganglioside-binding fragment and the H _N -domain of the Clostridia neurotoxin. 5. The fusion polypeptide of claim 4 wherein the linker is a GS20 linker. 6. Hybrid polypeptide according to any one of paragraphs. 2-5, where the activation site is an enterokinase activation site. 7. Hybrid polypeptide according to any one of paragraphs. 1-6, where the selective ganglioside-binding fragment contains amino acid residues 22-124 of SEQ ID NO: 9. 8. Hybrid polypeptide according to any one of paragraphs. 1-7 where: the light chain of Clostridial neurotoxin (L) contains amino acid residues 1-448 of SEQ ID NO: 1; H_N-clostridia neurotoxin domain contains amino acid residues 449-872 SEQ ID NO: 1; H_CN-clostridia neurotoxin domain contains amino acid residues 873-1094 SEQ ID NO: 1; as well as H_CC-clostridia neurotoxin domain contains amino acid residues 1095-1296 SEQ ID NO: 1. 9. Nucleotide sequence encoding a hybrid polypeptide according to any one of paragraphs. 1-8. 10. An expression vector containing the nucleotide sequence according to claim 9. 11. A cell for the expression of a hybrid polypeptide, containing the expression vector according to claim 10.

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