阅读说明：本技术 肉毒神经毒素生物杂化体 (Botulinum neurotoxin biohybrid ) 是由 帕尔·斯滕马克 杰弗里·马叙耶 于 2019-02-21 设计创作，主要内容包括：本发明涉及适合于协同结合至突触结合蛋白(Syt)受体、突触相关囊泡蛋白2(SV2)受体和神经节糖苷(Gang)受体的新的肉毒神经毒素(BoNT)重链结合结构域(H<Sub>C</Sub>/TAB)以及包含所述新的H<Sub>C</Sub>/TAB的多肽、编码所述多肽的载体及其用途。(The present invention relates to novel botulinum neurotoxin (BoNT) heavy chain binding domains (H) suitable for cooperative binding to the synaptic binding protein (Syt) receptor, synaptic associated vesicular protein 2(SV2) receptor, and ganglioside (Gang) receptor C TAB) and H comprising said new C Polypeptide of/TAB, vectors encoding said polypeptide and uses thereof.)

1. Having an N-terminus (H)_CN) And C terminal (H)_CC) Botulinum neurotoxin (BoNT) heavy chain binding Domain (H)_C/TAB), wherein said H_Cthe/TAB comprises:

a) a synaptotagmin (Syt) receptor binding site, and

b) a synapse-associated vesicular protein 2(SV2) receptor binding site, and

c) a ganglioside (Gang) receptor binding site,

and wherein said H_Cthe/TAB is adapted to coordinate binding to the synaptotagmin (Syt) receptor, the synaptotagmin 2(SV2) receptor and the ganglioside (Gang) receptor.

2. H according to claim 1_C/TAB, wherein the sequence forming the Gang receptor binding site is derived from any Gang receptor binding BoNT serotype and their subtypes.

3. H according to any one of claims 1 or 2_C/TAB, wherein the sequences forming the Syt receptor binding site are derived from any Syt receptor binding BoNT serotype and their subtype.

4. H according to any one of claims 1 to 3_C/TAB, wherein the sequence forming the site of SV2 receptor binding is derived from any SV2 receptor-binding BoNT serotype and their subtypes.

5. H according to any one of claims 1 to 4_C/TAB, wherein said H_CNThe sequences are derived from any SV2 receptor-binding BoNT serotype and their subtypes.

6. H according to any one of claims 1 to 5_C/TAB, characterized in that said H_CCThe domains interchangeably consist of sequences from BoNT serotype A (BoNT/A) and BoNT serotype B (BoNT/B).

7. H according to any one of claims 1 to 6_C[ TAB ] characterized by a sequence according to A₁B₁A₂B₂A₃Make up of the H_CCAnd (B) a sequence derived from BoNT/A, wherein A represents a sequence derived from BoNT/A and B represents a sequence derived from BoNT/B.

8. H according to claim 7_C/TAB, wherein B₁、A₂And B₂The sequence of (a) comprises mutations and/or deletions in order to address the entire H_CTAB produces a stable intramolecular interface.

9. H according to any one of claims 1 to 8_C/TAB, wherein the sequence forming the binding site for the Gang receptor is derived from BoNT/B.

10. H according to any one of claims 7 to 8_C/TAB, wherein the sequence forming the binding site for the Gang receptor is located at B₂In (1).

11. H according to any one of claims 1 to 10_C/TAB, wherein the sequence forming the Syt receptor binding site is derived from BoNT B, DC or G.

12. H according to any one of claims 7 to 8 or 10_C/TAB, wherein the sequence forming the Syt receptor binding site is located at B₁And B₂In (1).

13. H according to any one of claims 1 to 12_C/TAB, wherein said H_CNThe sequence is derived from BoNT/A.

14. H according to any one of claims 7 to 8, 10 or 12_C/TAB, wherein the sequence forming the binding site of the SV2 receptor is located at H_CNNeutralization of H_CCA in (A)₁And A₃In (1).

15. H according to any one of claims 1 to 14_C/TAB having an amino acid sequence which has at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to the sequence shown in SEQ ID No. 1.

16. A composition comprising H according to any one of claims 1 to 15_CThe polypeptide of/TAB, bound directly or via a linker to any one or more other proteins, polypeptides, amino acid sequences or fluorescent probes.

17. The polypeptide of claim 16, wherein said polypeptide is a BoNT polypeptide (BoNT/TAB) characterized in that said H is excluded_CIn addition to/TAB, said BoNT/TAB comprises a heavy chain shift domain (H) in said polypeptide sequence_N) Light Chain (LC) and the Light Chain (LC) located between the LC and H_NA protease site of (a), wherein said H_NAnd said LCs are derived from any BoNT serum type a, B, C, D, DC, E, En, F, G or X and subtypes thereof, respectively and independently of each other.

18. The polypeptide of claim 17, further comprising any other protein, polypeptide, amino acid sequence, or fluorescent probe linked thereto, either directly or through a linker.

19. The polypeptide of any one of claims 17 or 18, wherein the protease site is an exoprotease site.

20. The polypeptide of any one of claims 16 to 19, having an amino acid sequence which has at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to a sequence set forth in any one of seq id No.3, seq id No.5, seq id No.6, seq id No.8, seq id No.10 or seq id No. 12.

21. A vector comprising a nucleic acid encoding H according to any one of claims 1 to 15_CTAB or a polypeptide according to any of claims 16 to 20.

22. The vector of claim 21, comprising a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to a sequence set forth in any one of seq id No.4, seq id No.7, seq id No.9, seq id No.11 or seq id No. 13.

23. The H of any one of claims 1 to 15_CTAB or a polypeptide according to any of claims 16 to 20 for use in a method of treatment or in a cosmetic method.

24. H for use according to claim 23_C/TAB or a polypeptide, wherein the therapeutic or cosmetic method is the treatment of weak and/or stiff muscles.

25. H for use according to any one of claims 23 or 24_CTAB or polypeptide, wherein the method of treatment is the treatment and/or prevention of a condition selected from the group consisting of neuromuscular disorders and muscle spasm disorders.

26. H for use according to any one of claims 23 to 25_CTAB or a polypeptide, wherein the condition is selected from: spasmodic vocalization disorder, spasmodic torticollis, laryngeal dystonia, oromandibular dystonia, glossomahialgia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasm and other language disorders, spastic colitis, nervous system disorderNeurogenic bladder, anal spasms, limb spasms, tics, tremors, bruxism, anal fissures, achalasia, dysphagia and other dystonias and other disorders characterized by involuntary movements of the muscle group, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, secretory disturbances, pain due to muscle spasms, headaches, athletic injuries and depression.

27. The polypeptide of claim 16 for use in a pharmacological test to investigate the effect of the protein, polypeptide, amino acid sequence or fluorescent probe on synaptic processes.

28. The H of any one of claims 1 to 15_CTAB for use as a vehicle for efficient transport of any protein, polypeptide, amino acid sequence or fluorescent probe bound thereto to the surface of a neuron.

29. Use of BoNT/TAB according to any one of claims 17 to 20 as a vehicle for efficient transport of any protein, polypeptide, amino acid sequence or fluorescent probe to neuronal cytosol using a toxin translocation system.

30. A pharmaceutical or cosmetic composition comprising H according to any one of claims 1 to 15_CTAB or a polypeptide according to any one of claims 16 to 20.

31. A kit of parts comprising a composition according to claim 30 and instructions for therapeutic administration of the composition.

32. A method of treating a condition associated with undesired neuronal activity, the method comprising administering to a subject a therapeutically effective amount of H according to any one of claims 1 to 15_CTAB or a polypeptide according to any of claims 16 to 20 or a pharmaceutical composition according to claim 30, thereby treating said condition, wherein said condition is selected fromA group consisting of: spasmodic vocalization disorders, spasmodic torticollis, laryngeal dystonia, oromandibular dysarthria, glossomalgia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity and other language disorders, spastic colitis, neurogenic bladder, anal spasms, limb spasms, tics, tremors, bruxism, anal fissures, achalasia, dysphagia and other dystonias and other disorders characterized by involuntary movements of the muscle groups, tears, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, secretory disorders, pain due to muscle spasms, headaches, motor impairment and depression, and dermatological or aesthetic/cosmetic conditions.

Technical Field

The present invention relates to Botulinum neurotoxin polypeptides (Botulinum neurotoxins) and in particular to chimeric Botulinum neurotoxin heavy chains.

Background

Botulinum neurotoxin (BoNT) is the most potent protein toxin known to man and is a rare cause of paralysis, botulism. The bacterial toxin family consists of eight serotypes, BoNT/A-G, and the recently described BoNT/X (Montal, 2010; Zhang et al, 2017). It is composed of a base, a cover and a coverAll share a common structure and are expressed as 150kDa proteins that are cleaved post-translationally into double-stranded molecules consisting of a light chain (LC, 50kDa) linked to a heavy chain (HC, 100kDa) by a single disulfide bond. The HC has two functional domains, the N-terminal translocation domain (H)_N) And C-terminal binding Domain (H)_C) While LC is responsible for intracellular catalytic activity. Bonts first recognize cholinergic nerve terminals through specific cell surface receptors and then are endocytosed in vesicles. The acidic endosomal environment results in a conformational change that causes LC translocation (also known as toxin translocation) within the cytosol. Then, the free catalytic domain, zinc-protease, can specifically target one of three neuronal SNARE (soluble N-ethylmaleimide sensitive factor connexin receptors): (iii) BoNT/A,/C and/E cleave SNAP-25; BoNT/B,/D,/F,/G and/X target VAMP (synaptophysin); BoNT/C cleaves syntaxin (Schiiavo et al, 2000; Zhang et al, 2017). These three proteins form complexes that mediate the fusion of synaptic vesicles with the plasma membrane (Sudhof and Rothman, 2009). Proteolysis of any SNARE inhibits exocytosis and thus neurotransmitter release, effectively resulting in the flaccid paralysis symptoms of botulism (Rossetto et al, 2014). The sequences of three functional domains have been previously described (Lacy DB et al, 1999). The catalytic domain consists of amino acids 1-437, the translocation domain consists of amino acids 448-872 and the binding domain consists of amino acids 873-1295, which are referred to as the BoNT/A sequence in Lacy DB et al. Since all BoNT serotypes and their subtypes are largely homologous, the location of the corresponding domains in any other serotype or subtype will be very similar.

The high potency of these toxins makes them extremely useful therapeutic agents in the treatment of an ever-increasing range of neuromuscular disorders, such as strabismus, cervical dystonia and blepharospasm, as well as other conditions involving acetylcholine release, such as hyperhidrosis (Chen, 2012). BoNT/A and/B are the only serotypes that are approved and commercially available as therapeutics. BoNT/A is generally considered to be more potent in humans and is therefore in most cases the serotype of choice (Bentivoglio et al, 2015). However, treatment by BoNT usually requires repeated injections, since the therapeutic effect of the toxin is only transient. This is reported to result in tolerance in a small subset of patients who develop an immune response to BoNT/a (Lange et al, 2009; Naumann et al, 2013). Although BoNT/B represents an alternative, its lower potency means that higher doses are required and therefore represents a greater risk of immunogenicity (Dressier and Bigalke, 2005). In addition, BoNT/B is also associated with several adverse outcomes, such as injection pain, short duration of action, and more common side effects (Bentivoglio et al, 2015). The major side effects are often also associated with the treatment of muscle spasms, rather than cosmetic applications. This is because the side effects are essentially due to the diffusion of the toxin to other areas of the body, and the possibility of toxin diffusion is directly related to the injected dose. Side effects range from transient non-severe events, such as ptosis and diplopia, to life threatening events, and even death.

The binding of BoNT/a and/B to neurons has been characterized in detail and is based on a dual receptor mechanism, including synaptobrevin and gangliosides anchored on the neuronal membrane. The protein receptor for BoNT/A was identified as SV2(Dong et al, 2006; Mahhrhold et al, 2006). More precisely, BoNT/a can bind to several human SV2 isoforms A, B and C, although the toxin recognizes only the N-glycosylated form formed by SV2A and SV2B (Yao et al, 2016). The protein receptor for BoNT/B is synaptotagmin (Syt) (Nishiki et al, 1994, 1996; Dong et al, 2003), where Syt1 is preferred over Syt2 in humans (Strotmeier et al, 2012). For all bonts, ganglioside recognition is the first step of the intoxication process (Binz and Rummel,2009) and is mediated by a common binding mechanism in their sequence that is concentrated in the conserved motif h. BoNT/A binds preferentially to the terminal N-acetylgalactosamine-galactose moiety of GT1B and GD1a (Takamizawa et al, 1986; Schenggrund et al, 1991), while the data for BoNT/B suggest that the double salivary motif of GD1B and GT1B is preferred. Different serotypes differ in their carbohydrate specificity and affinity (Rummel, 2013).

The modular arrangement and unique properties of the various BoNT serotypes make toxins the target of choice for protein engineering. In particular, several studies have shown that it is possible to exchange the entire domain between serotypes (Masuyer et al, 2014) and thus obtain new toxins with unique drug potential. For example, several molecules have been generated that consist of a BoNT/B binding domain and a BoNT/A catalytic domain that are involved in translocation (Rummel et al, 2011; Wang et al, 2012; Kutschenko et al, 2017). These so-called chimeric toxins show attractive pharmacological properties in terms of potency and duration of activity, which, compared to SV2, are associated with high affinity of BoNT/B for synaptotagmin and high expression of this receptor on neurons (Takamori et al, 2006; Wilhelm et al, 2014).

Disclosure of Invention

Since both the production of neutralizing antibodies and toxin diffusion are directly related to the injected dose, it is highly desirable to reduce the toxin dose while maintaining the same level of toxin activity, which means that the potency of each toxin molecule must be increased. It is therefore an object of the present invention to increase BoNT polypeptides with improved duration and efficacy and with a lower risk of diffusion from the injection site. The present inventors have recognized key issues in previous work mentioned above in engineering chimeric BoNT polypeptides. None of the previous work has considered structural aspects of the polypeptide.

Using structure-based methods and current knowledge of the receptor binding mechanisms of BoNT/A and/B, the present inventors engineered a novel molecule, TriRecaBTox (BoNT/TAB), comprising a specifically engineered H capable of recognizing the SV2C receptor, the synaptotagmin receptor, and the ganglioside receptor_CDomain (H)_C[ TAB ]). The inventors have shown that BoNT/TAB can be expressed recombinantly and purified. Using X-ray crystallography, the inventors further demonstrated that BoNT/TAB can bind to its three receptors simultaneously. Therefore, BoNT/TAB should recognize neuronal cells with high affinity and have the potential to be a highly effective alternative to BoNT/A therapy.

Thus, by providing in a first aspect a botulinum neurotoxin (BoNT) heavy chain binding domain (H)_C/TAB) achieves the above object, wherein H_CTAB comprising a) a synaptotagmin (Syt) receptor binding site and b) A synapse-associated vesicular protein 2(SV2) receptor binding site and c) a ganglioside (Gang) receptor binding site, and wherein said H_Cthe/TAB is adapted to coordinate binding to the synaptotagmin (Syt) receptor, the synaptotagmin 2(SV2) receptor and the ganglioside (Gang) receptor.

H_C/TAB has an N-terminus (H)_CN) And C terminal (H)_CC). According to one embodiment, H_CCThe domains interchangeably consist of sequences from BoNT serotype A (BoNT/A) and BoNT serotype B (BoNT/B).

According to other embodiments, according to sequence A₁B₁A₂B₂A₃Make up of the H_CC-terminal, wherein A represents a sequence from BoNT/A and B represents a sequence from BoNT/B.

According to other embodiments, B₁、A₂And B₂The sequence of (a) comprises mutations and/or deletions in order to address the entire H_CTAB produces a stable intramolecular interface.

According to other embodiments, the sequences forming the Gang receptor binding site are derived from any of the Gang receptor-binding BoNT serotypes and subtypes thereof.

According to other embodiments, the sequence forming the Gang receptor binding site is derived from BoNT/B.

According to other embodiments, the sequence forming the binding site for the Gang receptor is located at B₂In (1).

According to other embodiments, the sequences forming the Syt receptor binding site are derived from any Syt receptor binding BoNT serotype and their subtypes.

According to other embodiments, the sequences forming the Syt receptor binding site are derived from BoNT B, DC or G.

According to other embodiments, the sequence forming the Syt receptor binding site is located at B₁And B₂In (1).

According to other embodiments, H_CNThe sequence is derived from any SV2 receptor-binding BoNT serotype and their subtypes.

According to other embodiments, H_CNThe sequence is derived from BoNT/A.

According to other embodiments, the sequence forming the SV2 receptor binding site is located at H_CNIs in H_CCA in (A)₁And A₃In (1).

According to other embodiments, H_C/TAB has an amino acid sequence which has at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity to a sequence set forth in any one of SEQ ID No.1, 3, 5, 6, 8, 10 or 12.

According to a second aspect, there is provided a kit comprising H according to the first aspect and any embodiment of the first aspect bound directly and via a linker to any other protein, polypeptide, amino acid sequence or fluorescent probe_C/TAB。

According to an embodiment of the second aspect, said polypeptide is a BoNT polypeptide (BoNT/TAB), characterized in that said BoNT/TAB is H-deleted_CIn addition to/TAB, a heavy chain translocation domain (H) is included in the polypeptide sequence_N) Light Chain (LC) and between LC and H_NA protease site arranged therebetween, wherein said H_NAnd LC are derived from any of BoNT sera type a, B, C, D, DC, E, En, F, G, or X and their subtypes, and BoNT-like polypeptides, respectively and independently of each other.

According to other embodiments, the polypeptide may comprise any other protein, polypeptide, amino acid sequence or fluorescent probe linked thereto, either directly or through a linker.

According to other embodiments, the protease site is an exoprotease site. According to other embodiments, the exoprotease site is a factor Xa site.

According to other embodiments, the polypeptide according to the second aspect has an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to a sequence set forth in any one of seq id No.1, 3, 5, 6, 8, 10 or 12.

According to a third aspect, there is provided a method comprising encoding H according to the first aspect and any embodiment of the first aspect_C/TAB, or a nucleic acid sequence of a polypeptide according to the second aspect and any embodiment of the second aspect.

According to a fourth aspect, there is provided H according to the first aspect and any embodiments of the first aspect_CUse of/TAB or a polypeptide according to the second aspect and any embodiment of the second aspect in a method of treatment or in a cosmetic method.

According to one embodiment of the fourth aspect, the method of treatment or cosmetic method is the treatment of weak and/or rigidified muscles (dampen and/or inactivated muscles).

According to other embodiments of the fourth aspect, the method of treatment is the treatment and/or prevention of a disorder selected from the group consisting of a neuromuscular disorder, a condition involving acetylcholine release, and a muscle spasm disorder.

According to other embodiments, the disorder is selected from: spasmodic vocalization disorder, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia (oromandibular dysphonia), glossomalgia, cervical dystonia, focal dystonia (focal hand dystonia), blepharospasm, strabismus, hemifacial spasm, blepharospasm, cerebral palsy, focal spasm and other language disorders, spastic colitis, neurogenic bladder, anal spasm, limb spasm, tics (tics), tremor, bruxism, anal fissure, achalasia, dysphagia and other dystonias well as other disorders characterized by involuntary movements of the muscle group, tearing, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, secretory disorders, pain due to muscle spasm, headache, motor impairment and depression.

According to other embodiments, H according to the first aspect and any embodiments of the first aspect_Cthe/TAB or the polypeptide according to the second aspect and any embodiment of the second aspect may be used in a pharmacological test to investigate the role of the protein, polypeptide, amino acid sequence or fluorescent probe in synaptic processes.

According to other embodiments, H according to the first aspect and any embodiments of the first aspect_Cthe/TAB or the polypeptide according to any embodiment of the second aspect and the second aspect may be used as any protein, polypeptide, amino acid sequence or fluorescence to be bound theretoThe probe is effectively transported to the medium on the surface of the neuron.

According to other embodiments, H according to the first aspect and any embodiments of the first aspect_Cthe/TAB or the polypeptide according to any embodiment of the second aspect and the second aspect may be used as a vehicle for efficient transport of any protein, polypeptide, amino acid sequence or fluorescent probe into the cytosol of neurons using a toxin translocation system.

According to a fifth aspect, there is provided H including any of the embodiments according to the first aspect and the first aspect_C/TAB or a polypeptide according to the second aspect and any embodiment of the second aspect.

According to an embodiment of the fifth aspect, the composition may further comprise a pharmaceutically and/or cosmetically acceptable excipient, carrier or other additive.

According to a sixth aspect, there is provided a kit of parts comprising a composition according to the fifth aspect and instructions for the therapeutic administration of said composition.

According to a seventh aspect, there is provided a method of treating a condition associated with unwanted neuronal activity, the method comprising administering to a subject a therapeutically effective amount of H according to the first aspect and any embodiment of the first aspect_C/TAB, or a polypeptide according to the second aspect and any embodiment of the second aspect, or a composition according to the fifth aspect, thereby treating the condition, wherein the condition is selected from the group consisting of: spasmodic vocalization disorder, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia (oromandibular dysphonia), glossomalgia, cervical dystonia, focal dystonia (focal hand dystonia), blepharospasm, strabismus, hemifacial spasm, blepharospasm, cerebral palsy, focal spasm and other language disorders, spastic colitis, neurogenic bladder, anal spasm, limb spasm, tics (tics), tremor, bruxism, anal fissure, achalasia, dysphagia and other dystonia and other disorders characterized by involuntary movements of the muscle group, tearing, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, secretion disordersPain due to muscle spasm, headache, sports injuries and depression, and dermatological or aesthetic/cosmetic conditions.

Drawings

FIG. 1: structural information of receptor binding by BoNT/A and/B. (a) Superposition of crystal structures of binding domains of BoNT/a complexed with GT1b (PDB2VU) and SV2C (PDB 5JLV) glycosylated with human. (b) Crystal structure of binding domain of BoNT/B in complex with GD1a and rat synaptotagmin 2(PDB 4 KBB). Representing proteins in a ribbon pattern and carbohydrates in a stick, (c) H_CSequence alignment of/A (Uniprot P10845) and/B (Uniprot P10844), where secondary structural elements are also provided (mapping by ESPript 3.0; Robert and Gouet, 2014). For each domain, the region directly involved in receptor binding is highlighted, with H for the SV2 receptor_CLines above the/A sequence are highlighted, and for the Syt receptor, as H_CLine below the/B sequence is highlighted; the ganglioside receptor binding site is underlined in striped gray line.

FIG. 2: having a passage H_CReceptor-bound H of/A and/B_CAlignment of/TAB. The protein sequences were aligned by ClustalO (Sievers et al, 2011). Highlighted in black (white) and light gray (black), respectively, at H_CH used in TAB design_CA and H_CSegment of/B. The positions in which the deletions are included are shown in dark grey (dashed lines).

FIG. 3: h_CCharacterization of/TAB. (a) Purified H_CSDS-PAGE analysis of/TAB and interaction with H_CA and H_CComparison of control/B (B) immunoblot analysis of the same samples as in (a) using a polyhistidine probe. "M" represents a molecular weight marker.

FIG. 4: x-ray crystal structure of the binding domain of treecabtox complexed with SV2C, human synaptotagmin 1 and GD1 a. (a) H_CTAB with ribbon representations of SV2C, hSyt1, and GD1 a. (b-d) SV2C receptor binding site (b), GD1a (c), and 2F at 2 σ around hSyt1(d)₀-F_cAn example of an electron density map (mesh).

FIG. 5: with the SV2 receptorAnd (4) combining. (a) H complexed with hSV2C_C[ TAB ] and H_CSuperposition of the crystal structure of/A (PDB 4 JRA). (b) H complexed with glycosylated hSV2_C[ TAB ] and H_CSuperposition of the crystal structure of/A (PDB 5 JLV). Residues involved in binding are shown as sticks (Benoit et al, 2014) and are according to the corresponding H_Cthe/A position is labeled.

FIG. 6: binding to synaptotagmin. H complexed with human Syt1 and rat Syt2, respectively_C[ TAB ] and H_CSuperposition of the crystal structure of/B (PDB 4 KBB). Residues involved in binding are shown as sticks (Jin et al, 2006; Chai et al, 2006) and are according to the corresponding H_Cthe/B position is labeled.

FIG. 7: in combination with GD1 a. H complexed with GD1a_C[ TAB ] and H_CSuperposition of the crystal structure of/B (PDB 4KBB) (shown as dark and light grey, respectively). Residues involved in binding are shown as sticks (Bernstson et al, 2013) and are according to the corresponding H_Cthe/B position is labeled.

FIG. 8: characterization of BoNT/TAB. (a) SDS-PAGE analysis of purified BoNT/TAB and correlation with H_CA and H_CComparison of the/B control. (b-d) using a polyhistidine probe (b); h_CA (c) and H_C(b) immunoblot analysis of antisera. The sample was the same as in (a), "M" represents a molecular weight marker.

FIG. 9: activation of BoNT/TAB. (a) Schematic of BoNT/TAB constructs depicting domain organization. Engineered protease activation sites are shown as black dashes. The natural disulfide bond between the light and heavy chains is shown as a black straight line. (b) SDS-PAGE analysis of BoNT/TAB activation assay. Non-reduced (NR) and reduced (R) unactivated BoNT/TAB (left panel) and factor Xa-activated BoNT/TAB (right panel), respectively. Annotating the segment of interest; "M" represents a molecular weight marker.

FIG. 10: h_CExtended use of/TAB. (a) And H_CSchematic of potentially functional BoNT derivatives related to TAB. The constructs will include a functional BoNT domain ("n") from any serotype or subtype. Protease activation sites (black dashed lines) should also be included. (b) Using H_CTAB will carry the thing eggSchematic representation of potential constructs for transport of leukocytes (nogo proteins) to neuronal cell surfaces.

FIG. 11: h_CPurification of TAB. (a) Chromatogram obtained by affinity chromatography purification Using 5ml HisTrap FF column (A)₂₈₀Traces). (b) Chromatogram obtained by size exclusion chromatography purification using a Superdex200 column (A)₂₈₀Traces). Highlighting the stages of the purification process and having H_CFraction of TAB.

FIG. 12: h complexed with SV2C, hSyt1 and GD1a_CCrystal of/TAB. (a) Crystals grown in 20% v/v polyethylene glycol 6000, 0.1M citrate pH 5.0. (b) Crystals fixed on cryo-loop for data collection at Diamond 104-1 workstation. (c) X-ray diffraction pattern of the crystal.

FIG. 13: purification of BoNT/TAB. (a) Chromatogram obtained by affinity chromatography purification Using 5ml HisTrap FF column (A)₂₈₀Traces). (b) Chromatogram obtained by size exclusion chromatography purification using a Superdex200 column (A)₂₈₀Traces). The stage of the purification process and the fractions with BoNT/TAB are highlighted.

FIG. 14: h complexed with SV2C, human synaptotagmin 1 and GD1a_CX-ray crystal structure of binding domain of/TAB. (a) And (b) H_C[ TAB ] (a) and H_CTemperature fragmentation analysis of the crystal structure of/TAB 2.1(B) -Putty radius representation, where the radius is proportional to the B-factor. Showing a ring "360", highlighting locations 360 and 362 in black, (c) H_CX-ray crystal structure of complex of/TAB 2.1 with SV2C, hSyt1 and GD1a (stick model representation), (d) same as (c) wherein the lipid binding loop is labeled and shows hydrophobic residues as stick model.

FIG. 15: h_CPurification of/TAB 2.1. (a) Chromatogram obtained by affinity chromatography purification Using 5ml HisTrap FF column (A)₂₈₀Traces), (b) chromatograms by gel filtration using a Superdex200 column (A)₂₈₀Traces). Shows to have H_CFraction of/TAB 2.1, (c) and (d) H_CCharacterization of/TAB 2.1. From purified H_C(ii)/TAB 2.1, SDS-PAGE fractions from affinity chromatography and gel filtration fractions from (c) and (d)Separating out; the first lane on the left shows molecular weight markers. Shows that corresponds to H_CTape of/TAB 2.1.

FIG. 16: h_C/TAB2.1.1 and H_CPurification of/TAB2.1.3. (a) And (b) are each from H_CAffinity chromatography purification and gel filtration chromatogram of/TAB2.1.1 (A)₂₈₀Traces). Shows to have H_CFraction (c) and (d) from H_CAffinity chromatography purification and gel filtration chromatogram of/TAB2.1.3 (A)₂₈₀Traces). Shows to have H_CFraction of/TAB2.1.3, (d) H_C/TAB2.1.1 and H_CCharacterization of/TAB2.1.3. SDS-PAGE analysis of the purified samples; the first lane on the left shows molecular weight markers. Shows that corresponds to H_C/TAB2.1.1 and H_CA strip of/TAB2.1.3.

FIG. 17: fig. X4: and (5) purifying the BoNT/TAB2.1.3. (a) And (b) SDS-PAGE analysis of fractions from BoNT/TAB2.1.3 purification. Fractions from affinity chromatography (a) and gel filtration (b); the first lane on the left shows molecular weight markers. Bands corresponding to BoNT/TAB2.1.3 are shown, (c) SDS-PAGE analysis of purified BoNT/TAB2.1.3 samples. In lane 1: sample before thrombin activation, in lane 2: final activated samples (after thrombin treatment). Bands corresponding to full-length (single-stranded) HC and LC are shown. The right lane shows the molecular weight markers, (d) chromatogram from the final gel filtration chromatography (after thrombin cleavage) using a Superdex200 column (A)₂₈₀Traces). Fractions with BoNT/TAB2.1.3 are shown.

Definition of

As used herein, the term botulinum neurotoxin "BoNT" encompasses any polypeptide or fragment from a botulinum neurotoxin. The term BoNT may refer to full-length BoNT. The term BoNT may refer to a BoNT fragment that may implement an overall cellular mechanism whereby BoNT enters neurons and inhibits neurotransmitter release. The term BoNT may simply refer to a BoNT fragment without requiring that the fragment have any particular function or activity.

As used herein, the term "translocation domain" or "H_N"indicates that mediation of BoNT light chain can be effectedBoNT domain of shift step of shifted poisoning process. Thus, H_NFacilitating movement of the BoNT light chain across the membrane into the cytoplasm of the cell.

As used herein, the terms "binding domain" and "H_CDomain "is synonymous and means any naturally occurring BoNT receptor binding domain that can perform the cell binding step of a poisoning process, including, for example, the binding of BoNT to a BoNT-specific receptor system located on the plasma membrane surface of a target cell.

In the present disclosure, the terms "nucleic acid" and "gene" are used interchangeably to describe a nucleotide sequence or polynucleotide encoding a polypeptide.

Detailed Description

As explained above in the background section, BoNT comprises a Light Chain (LC) linked to a Heavy Chain (HC) by a single disulfide bond. The Heavy Chain (HC) has two functional domains, the N-terminal translocation domain (H)_N) And C-terminal binding Domain (H)_C) While LC is responsible for intracellular catalytic activity. Thus, H_CComprising a receptor binding domain capable of specifically and irreversibly binding to a specific receptor expressed on a sensitive neuron, whereas H_NA channel is formed that allows the translocation of the attached LC from the endosomal-like membrane vesicle to the cytosol. The different BoNT serotypes have a locus H_CAnd (c) a different set of receptor binding sites, typically two receptor binding sites. The present inventors have used this knowledge to engineer novel BoNT H's containing binding sites for three different receptors_CBinding Domain (H)_C/TAB)。

The inventor designs H through engineering_Cthe/TAB domain achieves this object, said H_Cthe/TAB field contains:

a) a synaptotagmin (Syt) receptor binding site, and

b) a synapse-associated vesicular protein 2(SV2) receptor binding site, and

c) ganglioside (Gang) receptor binding sites.

Engineering of H_CStructure of the/TAB Domain allowed H_CSynergistic binding of/TAB to synaptotagmin (Syt) receptor, synaptically associated vesicle proteinThe white 2(SV2) receptor and the ganglioside (Gang) receptor. Thus, synergistic binding to three receptors on neuronal cells was achieved, resulting in binding to other BoNT H_CDomain comparison, new H_Cthe/TAB domain has an increased affinity. Thus, overall binding to neurons is improved and thus the efficacy of the toxin is improved.

H_CFurther comprising an N-terminus (H)_CN) And C terminal (H)_CC). An important feature of the present invention is that H_CH of/TAB_CCTerminal structure, which is the position of the receptor binding domain in BoNT.

At H_CIn one embodiment of/TAB, H_CCEnds interchangeably consisted of sequences from BoNT serotype A (BoNT/A) and BoNT serotype B (BoNT/B). By engineering the interchangeable structure, the inventors have been able to optimize the synergistic binding to all three receptors.

In other embodiments of the invention, according to sequence A₁B₁A₂B₂A₃Composition H_CCEnd, where A represents the sequence from BoNT/A and B represents the sequence from BoNT/B, see FIG. 2. This further optimizes H_CStructure of/TAB to allow cooperative binding of three receptor binding domains to at least all three of said receptors, possibly even simultaneously. The inventors have shown that by this A₁B₁A₂B₂A₃Sequence, simultaneous binding to all three receptors occurs in vitro. Engineered A according to this embodiment is depicted in SEQ ID No.1₁B₁A₂B₂A₃And (4) sequencing.

In order to further optimize H according to the above_C/TAB, mutations and deletions have been introduced to create a stable intramolecular interface, see, FIG. 2. In seq.id.no.1, substitutions were made in positions 306, 360 and 362 and deletions were made between positions 265/266 and 360/361 compared to the original sequence. However, the skilled person will understand that mutations in amino acids at positions +1, +2, +3, +4, +5 or-1, -2, -3, -4 or-5 from the positions indicated aboveAnd/or the deletion may have the same effect. Thus, any such modification at a position +/-5 amino acids from the indicated amino acid position is within the scope of the present disclosure.

According to the above specific embodiments and all of the following examples, the ganglioside receptor binding site is derived from BoNT/B, but it is envisaged that it may be derived from any Gang receptor-binding BoNT serotype and their subtypes, such as BoNT sera type a, B, C, D, DC, E, En, F, G or X or subtypes thereof, as all serotypes have a ganglioside receptor binding site.

According to a preferred embodiment of the invention, the sequence forming the binding site for the Gang receptor is located at B₂In (1).

The SV2 receptor binding domain may be derived from any SV2 receptor-binding BoNT serotype in general and their subtype, and in particular from BoNT serum A, D, E and type F. In the above detailed description and all following examples, the SV2 receptor binding domain was derived from BoNT/a, but as the skilled person will appreciate, H according to the appended claims_CFor purposes and intended use, any serotype comprising the SV2 receptor binding domain may be used as the source of the domain.

Part of the SV2 receptor binding domain is present in H_CNAnd (4) an end. Thus, H_CNThe sequence may be derived from any SV2 receptor-binding BoNT serotype and their subtypes. In the foregoing detailed description and all of the following examples, H_CNThe end is derived from BoNT/A. However, as the skilled person will appreciate, H is H as long as the SV2 receptor binding domain is functional_CNThe sequence may also be derived from any BoNT serum C, D, E, F or type G.

Furthermore, in accordance with a preferred embodiment of the present invention, the sequence forming the SV2 receptor binding site is located at H_CNNeutralization of H_CCA in (A)₁And A₃In (1).

The Syt receptor binding site may be derived from any Syt receptor binding BoNT serotype and their subtypes. Specifically, the Syt receptor binding site may be derived from BoNT serogroup B, serochimeric DC, or serogroup G. Advantages according to the inventionIn an alternative embodiment, the sequence forming the binding site for the Syt receptor is located at B₁And B₂In (1).

The invention also provides a composition comprising H according to the above_CA polypeptide of TAB. Thus, the polypeptide may comprise a linker directly or via a linker to H_CTAB-binding any other protein, polypeptide, amino acid sequence or fluorescent probe. Hereinafter, with H_Cthe/TAB-binding protein, polypeptide or amino acid sequence is referred to as "protein".

According to a preferred embodiment, said polypeptide is a recombinant BoNT polypeptide (BoNT/TAB) further comprising H in the polypeptide sequence_NAnd LC and at LC and H_NAn exoprotease site in between.

The exoprotease site enables the single chain polypeptide to be cleaved into a double-stranded molecule, thereby making the molecule an active toxin. According to an embodiment of the invention, the exoprotease site is a factor Xa site, although this is not a limiting feature of the polypeptide according to the invention.

According to one embodiment, BoNT/TAB in its active form is according to seq.id No. 5. According to another embodiment, BoNT/TAB in its active form is according to any one of the sequences shown in seq.id No.6, 8, 10 or 12. Preferably, BoNT/TAB in its active form is according to seq.id No. 12.

Said H_NAnd LC can be derived from any BoNT serum type a, B, C, D, DC, E, En, F, G, or X and their subtypes, as well as BoNT-like polypeptides, separately and independently. New proteins similar to BoNT, i.e. with similar collar structure and varying degrees of sequence identity, but produced by other organisms than clostridium botulinum (c. Thus, the skilled person will be able to select H from any BoNT serotype, subtype thereof or BoNT-like polypeptide_NAnd/or LC.

As indicated above, H is introduced_CMutations and deletions in/TAB further ensure that engineered BoNT/TAB can be generated as soluble proteins with the correct structure and desired activity.

Preferably, as required, heavyGroup generated H_C/TAB, the polypeptide according to which has been produced recombinantly.

Accordingly, the present disclosure also provides for encoding any of the H's according to the above_CIsolated and/or recombinant nucleic acids of TAB or polypeptides. Encoding H as described in the disclosure of the invention_CThe nucleic acid for/TAB or polypeptide may be double-stranded or single-stranded DNA or RNA. In certain aspects, a test nucleic acid encoding an isolated polypeptide fragment is also understood to comprise a nucleic acid encoding a polypeptide as any of the H's described herein_CA nucleic acid of a polypeptide of TAB or a variant of the polypeptide. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.

The invention also provides a method comprising encoding H according to the above_CA vector comprising the nucleic acid sequence of TAB. The vector may further comprise a coding sequence and H_CTAB together with any other protein or probe produced recombinantly, thereby obtaining H in one polypeptide_CThe protein or probe to which TAB binds. The vector is preferably an expression vector. The vector may comprise a promoter operably linked to the nucleic acid. A variety of promoters can be used for expression of the polypeptides described herein, and they are known to those of skill in the art.

The expression vector comprising the nucleic acid can be transferred to a host cell by conventional methods (e.g., electroporation, lipofection, and calcium phosphate precipitation), and the transfected cell can then be cultured by conventional methods to produce the polypeptide described herein. In some embodiments, expression of a polypeptide described herein is regulated by a constitutive, inducible, or tissue-specific promoter.

The polypeptide may be produced in any eukaryotic or prokaryotic cell, or in yeast. The polypeptides according to the invention can also be produced in a cell-free system. The skilled person will readily be able to apply the selected expression system to humans. The expression system used to produce the polypeptides of the invention is not limited in scope by the invention.

Purification and modification of recombinant proteins are well known in the art, and thus the design of a polymeric protein precursor may include some embodiments as readily understood by the skilled artisan.

The protein to be included in the polypeptide may be any protein of interest to be transported to and/or internalized into a neuronal cell.

If internalization of the protein is desired, it may be advantageous to include H according to the description above in the polypeptide_NAnd H_CTAB, and replacement of LC with the protein of interest, since H_NA channel will be provided that allows the protein to translocate into the neuronal cell. If the neuronal cell surface is a protein target, it may be advantageous to bind the protein of interest directly to H_C[ solution ] TAB. Thus, based on the targeted delivery, the following combinations can be obtained:

i) protein-H_C/TAB

ii) protein-H_N-H_C/TAB

iii) protein-LC-H_N-H_C/TAB

According to the above i), by reacting the carrier protein with H_CThe binding of/TAB allows targeting of cargo proteins to neuronal surfaces. Some internalization by conventional cell surface recycling procedures will likely occur, but neuronal surface will be the primary target for this approach.

By binding a cargo protein to ii) and H according to above_CTAB-bound H_NOr bound to BoNT/TAB according to iii) above, the carrier protein can be transported more efficiently inside neurons using a toxin translocation system. As described in the background, once the BoNT toxin is internalized in neuronal cells in the vesicle, the acidic endosomal environment in the vesicle results in a conformational change that allows translocation of the LC from the vesicle to the cytosol of the cell. Thus, by using BoNT/TAB, the toxin translocation system, which is a mechanism for translocating the LC of BoNT from the internalization vesicle into the cytosol, can be used to translocate the above-described cargo protein into the cytosol of neuronal cells. The cargo protein can bind to H_NRather than LC, and as disclosed above, has a cargo protein and H_NIn between, or the carrier protein may bind to the LC. Both variants will enable the transport of cargo proteins to cells of neuronal cellsIn the liquid.

Thus, H_Cboth/TAB and BoNT/TAB can be used as vehicles for the transport of any protein to and/or into neurons. This also provides for the use of H in pharmacological testing_CThe possibility of/TAB and/or BoNT/TAB to study the role of proteins in, for example, synaptic processes.

The cargo protein may, for example, be any protein tag, such as an affinity or fluorescent tag or probe. Thus, any nucleic acid corresponding to such a protein tag may be included in the vectors disclosed above. The skilled person will be able to use standard cloning methods to include any gene of interest in the vector, as well as standard protocols for protein expression.

The binding domain of BoNT and the cargo protein can be expressed separately by a sortase system that allows their post-translational recombination. Thus, the transpeptidase activity of sortases can be used as a tool for the in vitro production of fusion proteins, and is well within the knowledge of those skilled in the art. Briefly, a recognition motif (LPXTG) is added to the C-terminus of the protein of interest, while an oligoglycine motif is added to the N-terminus of the second protein to be linked. Once sortase is added to the protein mixture, the two peptides are covalently linked by a natural peptide bond. The method may be used to produce a polypeptide according to the invention. In the context of the present invention, this would mean adding a recognition motif to the C-terminus of the protein of interest, while an oligoglycine motif is added to H_CThe N-terminus of/TAB or BoNT/TAB.

In addition, H can be used in therapeutic or cosmetic methods_C/TAB and/or BoNT/TAB. In general, H_CThe use of/TAB and/or BoNT/TAB may be very similar to the use already for BoNT/A and/or BoNT/B products. These include methods and treatments wherein the purpose of the methods and treatments is to weaken and/or rigidify a muscle.

H according to the invention_Cthe/TAB enables the injection of BoNT/TAB with higher affinity into cells and therefore higher efficiency. Thus, low doses are required and longer action times are possible. Thus, with BoNT/A or BoNT/BIn contrast, for the same effect, a smaller amount of BoNT/TAB can be injected, which reduces side effects, so less BoNT/TAB will diffuse from the injection site. With higher efficiency, stronger and more effective binding and lower required dose, the amount of excess BoNT/TAB available for diffusion beyond the injection site is less. Furthermore, BoNT can be administered less frequently with sustained effect, which will also minimize the risk of immune responses and adverse reactions and their consequences.

May be prepared by a reaction of H according to the above_CTypical medical conditions for treatment and/or prevention of/TAB and/or BoNT/TAB are disorders selected from the group consisting of: neuromuscular disorders, conditions involving the release of acetylcholine, and muscle spastic disorders. More specifically, it may relate to a condition selected from: spasmodic vocalization disorder, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia (oromandibular dysphonia), glossomalgia, cervical dystonia, focal dystonia (focal hand dystonia), blepharospasm, strabismus, hemifacial spasm, blepharospasm, cerebral palsy, focal spasm and other language disorders, spastic colitis, neurogenic bladder, anal spasm, limb spasm, tics (tics), tremor, bruxism, anal fissure, achalasia, dysphagia and other dystonias well as other disorders characterized by involuntary movements of the muscle group, tearing, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, secretory disorders, pain due to muscle spasm, headache, motor impairment and depression.

For cosmetic methods, H_Cthe/TAB and/or BoNT/TAB may preferably be used for preventing and/or treating wrinkles, frontal wrinkles or undesired lines to reduce said wrinkles, frontal wrinkles and lines.

H according to the above_Cthe/TAB and/or BoNT/TAB may be formulated into any suitable pharmaceutical or cosmetic composition. Comprising H_CThe pharmaceutical compositions of/TAB and/or BoNT/TAB may also contain pharmaceutically acceptable excipients, carriers or other additives. Comprising H_CThe cosmetic compositions of/TAB and/or BoNT/TAB may also contain cosmetically acceptable excipients, carriers, or other additives.

Administration of the pharmaceutical or cosmetic composition may be by injection, wherein the injection is administered at a site in the body where there is undesired neuronal activity. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If necessary, the composition may further comprise a solubilizing agent and a local anesthetic to relieve pain at the injection site.

In addition, the pharmaceutical or cosmetic composition may be contained in a kit with instructions for therapeutic administration of the composition. In such kits, the components of the compositions can be provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or anhydrous concentrate, in a closed container such as an ampoule or a bag (sachette) indicating the amount of active agent. The composition may be administered by infusion and, in this case, may be dispensed via an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration. Compositions for systemic administration may be liquids, for example, sterile saline, lactated ringer's solution or hank's solution. In addition, the compositions may be in solid form and redissolved or suspended immediately prior to use. Freeze-dried forms are also contemplated. The composition may be contained within lipid particles or vesicles, such as liposomes or microcrystals, which are also suitable for parenteral administration.

Thus, the present inventors have developed engineered BoNT biohybrids suitable for binding to all three of the SV2C receptor, the synaptotagmin receptor, and the ganglioside receptor simultaneously. Thus, BoNT biohybrids with higher potency, potency and duration than prior art BoNT polypeptides are provided. Thus, the use of the biohybrid of the invention enables the administration of lower doses of toxin than according to the prior art, while maintaining the same effect. Furthermore, the use of the biohybrid of the invention enables more frequent administration than previously used bonts. Thus, treatment of patients with BoNT biohybrids of the present invention will be more comfortable, wherein administration does not have to occur as frequently as in the prior art.

Experimental part

Materials and methods

Constructs. Will encode H_CAnd full-length (inactive) TriRecaBTox (H, respectively)_C/TAB and BoNT/TAB) for e.coli (e. coli) expression (see DNA sequence supplement) were codon optimized, synthesized and cloned into pET-28a (+) vector with an N-terminal 6 × His tag (GenScript, NJ, USA). The TriRecaBTox construct used in our study had three mutations at the catalytic site to avoid any safety issues (E224Q/R363A/Y366F) (Rossetto et al, 2001; Binz et al, 2002). The BoNT/TAB gene encodes 1311 amino acids, and H_Cthe/TAB gene corresponds to residue [875-1311 ]]。

Protein expression and purification. Plasmids with the genes of interest were transformed into E.coli (E.coli) BL21(DE3) cells (New England BioLabs, USA). Similar protocols were used for both proteins. In the LEX system (Epyphite3, Canada), expression was performed by growing cells in super broth with 50. mu.g/ml kanamycin at 37 ℃ for about 3 hours, then inducing with IPTG at a final concentration of 1mM, and maintaining overnight at 18 ℃. Cells were harvested and stored at-80 ℃. Cell lysis for protein extraction was performed by Emulsiflex-C3(Avestin, Germany) at 20kPsi in 25mM HEPES pH7.2 with 200mM NaCl, 25mM imidazole and 5% (v/v) glycerol. The cell debris was pelleted by ultracentrifugation at 267,000g for 45min at 4 ℃. The protein was first purified by affinity chromatography: the supernatant was loaded onto a 5ml HisTrap FF column (GE Healthcare, Sweden), washed with 25mM HEPES pH7.2, 200mM NaCl, 25mM imidazole and 5% (v/v) glycerol, and the protein was eluted with 25mM HEPES pH7.2, 200mM NaCl, 250mM imidazole and 5% (v/v) glycerol. The samples were then dialyzed overnight against 25mM HEPES pH7.2, 200mM NaCl and 5% (v/v) glycerol, and then subjected to a final size exclusion purification step using a Superdex200 column in a similar buffer (GE Healthcare, Sweden). H is to be_Cthe/TAB was maintained at 4.5mg/ml and BoNT/TAB at 7.3mg/ml in 25mM HEPES pH7.2 with 200mM NaCl, 0.025mM TCEP and 5% glycerol.

And (4) protein characterization. Protein samples were analyzed by gel electrophoresis using a NuPAGE 4-12% Bis-Tris gel and by immunoblotting performed on a PVDF membrane (ThermoFisher, Sweden). In-house preparation (in rabbits) of anti-H_CA and H_CFirst antibody of/B and detected with anti-rabbit IgG-peroxidase antibody (catalog # SAB3700852, Sigma, Sweden). Poly-histidine tags were detected using HRP-conjugated monoclonal antibodies (adi.1.10, catalog # MA1-80218, ThermoFisher, Sweden). TMB substrate (Promega, Sweden) was used for the detection. Comprises with H_CTAB similarly purified and His-tagged H_CA and H_CInternal control consisting of/B for comparison.

Activation of BoNT/TAB. A full-length (inactive) trerecabtox was designed for activation into a double-stranded form by a factor Xa cleavage site (IEGR) located between the light and heavy chains. Activation was performed by incubating 100. mu.g of BoNT/TAB with 2. mu.g of factor Xa (New England BioLabs, USA) overnight at 4 ℃. The activation results were analyzed by gel electrophoresis (as above).

Cloning, expression and purification of SV 2C-L4. The interacting part of the fourth luminal domain of synaptovesicle glycoprotein 2C was amplified from cDNA (SV2C-L4, residue 474-567, Uniprot ID Q.496J9) and cloned into pNIC28-Bsa4 (N-terminal His6 tag with TEV site) using LIC cloning. SV2CL4 was expressed in e.coli (e.coli) BL21(DE3) (New England BioLabs, USA) using a procedure similar to that described above. His-tagged SV2C-L4 was purified by affinity chromatography on a 2 mM LHISTrap HP column (GE Healthcare, Sweden) washed with 20mM HEPES, pH7.5, 500mM NaCl, 10% (v/v) glycerol, 50mM imidazole and 0.5mM TCEP. The protein was eluted with 20mM HEPES, pH7.5, 500mM NaCl, 10% (v/v) glycerol, 500mM imidazole and 0.5mM TCEP. SV2CL4 was then further purified by size exclusion chromatography using a Superdex75HiLoad 16/60 column (GE Healthcare, Sweden) in 20mM HEPES, pH7.5, 300mM NaCl, 10% (v/v) glycerol and 0.5mM TCEP.

X-ray crystallography. By adding 3.6mg/ml of H at room temperature_CTAB with 1mg/ml SV2C-L4 (recombinant human SV2C extracellular loop-4 [ residue 475-565)]) 1mM hSyt1 peptide (GEGKEDAFSKLKEKFMNELHK, by GenScript, USA, Inc.)Made) and 4mM GD1a oligosaccharide (Elicityl, France) were pre-incubated for 15min to prepare samples for crystallization.

Using a dropping (sitting drop) apparatus, crystals were grown by mixing 100nl of a 200nl sample of stock solution consisting of 20% v/v polyethylene glycol 6000, 0.1M citrate pH 5.0 (JCSG-plus screen B9, Molecular Dimensions, United Kingdom) and incubated at 21 ℃. Crystals appeared within 2 weeks and were transferred to a cryo-ring and frozen in liquid nitrogen.

Diffraction data was collected at workstation 104-1 equipped with a Diamond light source (Didcot, UK) with a PILATUS-6M detector (Dectiris, Switzerland). At 100 ℃ K, collect from the single crystal

The complete data set of (2). The raw data images were processed and scaled by DIALS (Gildea et al 2014) and AIMLESS (Evans 2006) using CCP4 suite 7.0(CCP4, 1994).

By complexing H with SV2C-L4_CA (PDB code 4JRA) and H complexed with rat SytII and GD1a_CModel prepared from coordinates of/B (PDB code 4KBB) was subjected to molecular replacement to determine the initial phase for structural analysis in PHASER (McCoy et al, 2007). The working model was refined using REFMAC5(Murshudov et al, 2011) and manually adjusted by COOT (Emsley et al, 2010). Water molecules are added at positions where the Fo-Fc electron density peak is greater than 3 σ, and potential hydrogen bonds may be added. Verification was performed by MOLPROBITY (Chen et al, 2010). Ramachandran statistics showed that 97.0% of all residues were in the most favorable region, with only one outlier in the unacceptable region. The crystallography statistics are summarized in table 1. By PyMOL: (LLC, USA).

Results

Design of TriRecaBTox: engineered botulinum toxin with three receptor binding sites

To realize the concept of a three-receptor toxin, the inventors first analyzed BoNT/A and/B for their receptor molecules in relation to each otherAnd structure information available for action. Recent work by Yao et al (2016) and Benoit et al (2014) provided X-ray crystal structures of the receptor binding domain of BoNT/a complexed with SV2C with (PDB 5JLV) and without post-translational modification (PDB 4JRA), respectively. The lumenal domain (loop 4) of SV2C forms a backbone-to-backbone interaction with H primarily_CA-bound quadrilateral beta-helix with open beta-strands at the interface of the two subdomains, while the N-glycan of SV2C faces H_CNExtended (fig. 1). Overall, these structures indicate that the common binding pattern with the two SV2 forms should also be extended to glycosylated SV2A and SV2B (Yao et al, 2016). These studies highlighted the key residues and multiple sites involved in the toxin-SV 2 interaction that should therefore be retained in the design of the trerecabtox (fig. 1). These include the BoNT/A segment [949-]、[1062-1066]、[1138-1157]And [1287-]. Residue numbering is based on the BoNT/A1 sequence (Uniprot-P10845).

Several crystal structures of BoNT/B complexed with synaptotagmin have also been described and help to define the interaction of the toxin with its receptor (Chai et al, 2006; Jin et al, 2006; Berntsson et al, 2013) (fig. 1). Once bound, the Syt peptide adopts a short helical structure that binds along a groove on the distal end of the C-terminal subdomain, which is directly involved in the segments [1113-1118] and [1183-1205] of BoNT/B. Residue numbering is based on the BoNT/B1 sequence (Uniprot-P10844). Therefore, these regions are considered necessary for inclusion in the trinecabtox construct.

In addition, the crystal structures of complex with their ganglioside receptors, BoNT/A and/B (Stenmark et al, 2008; Hamark et al, 2017; Berntsson et al, 2013), provide detailed descriptions of carbohydrate binding sites for each serotype. The site is highly conserved in the botulinum neurotoxin family and consists of H_CCThe shallow pocket on the subdomain (FIG. 1) consists of the central SxWY motif (1264-1267 in A; 1260-1263 in B) and the surrounding loop region. Notably, this pocket is close to the Syt receptor binding site in BoNT/B, which passes through loop [1244-1253 ]]Separately, however, allosteric effects have not been reported for simultaneous binding of two receptors (Bertnsson et al, 2013). To the maximum extentThe degree of any structural changes to the Syt receptor binding site was reduced, and it was thought that BoNT/B rather than the ganglioside receptor-binding site of BoNT/a would be more suitable for introduction in the design of trinecabtox.

After identifying components from the two serotypes necessary for binding to three different receptors, further structural analysis was performed to integrate them into a single molecule. To this extent, the primary sequences of BoNT/a (Uniprot P10845) and BoNT/B (Uniprot P10844) were aligned by ClustalO (Sievers et al, 2011) and the stereo structures of their binding domains were superimposed (fig. 1). Both serotypes share 40% overall sequence identity, however for H, which is the major region responsible for receptor recognition_CThe similarity was reduced to 34% for the C-terminal subdomain. The core fold of the binding domain is conserved across all clostridial neurotoxins (Swaminathan, 2011; Rummel et al, 2011), but the length of the connecting loops varies significantly. Therefore, it is important to also consider the secondary structure (fig. 1) in order to keep the major domain structure intact. Thus, the template of the newly designed molecule appears as multiple alternations between the BoNT/a and/B elements, creating new non-natural intramolecular interfaces that may be incompatible. To H_CA and H_CExamination of the superimposed crystal structure of/B enabled the inventors to optimize the design by correcting potential conflicts through single amino substitutions or deletions in critical positions (fig. 2). Specifically, the side chains of each residue within the collision region were examined, resulting in three substitutions in BoNT/B with equivalent BoNT/A amino acids: n1180, G1234, N1236(seq. id No. 3). In addition, several amino acids were removed (FIG. 2) to match secondary structural elements and to compensate for length differences between BoNT/A and/B in the loop regions of the transition interfaces (transitions interfaces). Deletions were made between L1139 and G1140 and between G1234 and T1235 (see SEQ. ID. No.3) compared to BoNT/A and BoNT/B sequences (FIG. 2).

The resulting molecule, designated as treecabtox, should be able to bind to three receptors: SV2, synaptotagmin, and gangliosides. The protein sequences are provided in seq.d. No.3 (inactive form) and seq.d. No.5 (active form).

Production and characterization of the TriRecaBTox binding domain.

The first step in TriRecaBTox characterization is the recombinant production of the binding domain (H)_C/TAB) to analyze its biochemical properties. For this purpose, the protein sequence was codon optimized for expression in e. The resulting gene was cloned into the pET-28a (+) vector to contain an N-terminal poly-histidine tag and facilitate the protein purification process, detailed information is provided in the methods section. The inventors showed that H (FIG. 3) can be expressed and partially purified using affinity chromatography and size exclusion techniques (FIG. 11)_C[ solution ] TAB. The original sample presents some low molecular weight contaminants that may correspond to residual host cell proteins. Other purification steps using methods such as ion exchange or hydrophobic interaction chromatography should help to obtain high purity samples. Confirmation of His-tagged H by immunoblotting_CThe presence of/TAB, where a single band was observed at the expected size (about 53kDa) (FIG. 3).

Crystal structure of the TriRecABTox binding domain complexed with its three receptors.

To evaluate H_CTAB ability to bind to its three receptors, a co-crystallization assay was established which included a fragment with the human SV2C luminal domain [ residues 475-565]Human Syt1 peptide [ residues 34-53 ]]And GD1a carbohydrate H_C[ solution ] TAB. High resolution diffraction is obtained

And a complete data set can be collected (table 1). The structure was resolved by molecular replacement using an input model with all potential components. The structure was resolved to confirm that the crystal structure contained all four elements: h binding to its three receptors simultaneously_C[ TAB ] (referred to as H)_C/TAB-3R) (FIG. 4). This result provides the first experimental evidence that trinecabtox can achieve its purpose in vitro, and it also enables a detailed overall analysis of the receptor binding mechanism at the atomic level. Using the newly determined structural information, we can directly compare H_C/TAB、H_C/A、H_CThe interaction between/B and their respective receptors.

Table 1. X-ray crystallography: data collection and refinement statistics

The values in parentheses are the values of the highest resolution shell.

First, the binding domain of the newly designed BoNT/TAB appears to have two subdomains: lectin-like H_CNAnd H_CCExpected folding of beta-clover fold (fig. 4). The resulting plurality of new intramolecular interfaces does not interfere with the overall structure, e.g. by reacting with H_CWhen A is superimposed

Low root mean square error (rmsd) (for 364 ca) and when compared to H_Cwhen/B is superimposedLow root mean square error (for 370 ca). Except for the aberrant N-terminal poly-histidine tag and loop [1169-]For complete H_CTAB modeling [876-]. The lack of electron density for these moieties can be explained by the fact that: these regions do not participate in any interactions and are located in solvent accessible regions of the crystal.

H is to be_CH of structure/TAB-3R complexed with SV2C_CStructure of/A. The structure of the SV2C luminal domain is identical in both complexes, with an rmsd of

(relative to 88 C.alpha.). Based on H_CDomain, three-dimensionally aligned two structures, and as expected by the inventors' design, showed SV2C at the same position (fig. 5). In particular, has been designated as being necessary for SV2 receptor binding and is contained in H_CFrom H in TAB_CThe region of/A is completely conserved. By passingPISA analysis H_CThe interface between/A and SV2C (Kissinel,2015) and which corresponds to a system comprising mainly electrostatic interactions

Wherein the open strands from the two proteins form complementary beta-sheet structures (Benoit et al, 2014). Using H_CThe corresponding analysis of TAB shows

Has SV2C and confirms a binding mechanism having a considerable number of hydrogen bonds. In addition, the inventor also prepares H_CTAB-3R and H_CThe potential binding to glycosylated SV2 was considered in comparison with the/A-gSV 2C complex (FIG. 5). Recently, N-glycosylation of N559 has been shown to be essential for receptor recognition and conserved among the SV2 isoforms (Yao et al, 2016). Notably, H, with or without glycosylation_CThe protein-protein interactions between/A and SV2C are highly similar. Carbohydrate chain towards H_CNThe subfields are extended. H involved in protein-glycan interactions_CAnalysis of the/A residues showed that they are located at H_CIs completely conserved in/TAB-3R, thus H_CTAB should recognize the N-glycosylated isoform of SV 2.

Then, the inventors convert H to_CStructure of/TAB-3R with H complexed with rSyt2_CThe structure of/B was compared. BoNT/B is expected to bind to human synaptotagmin a similar manner to its rodent homologues, despite the different affinities (Tao et al, 2017). In the crystal structures provided herein, hSyt1 also assumes a position H at H with rSyt2_CThe same binding groove in the alpha-helix structure in the/B (FIG. 6). To each of them H_CThe overlap of the hSyt1 and the rSyt2 of the domains confirms the conserved peptide configuration with rmsd as(relative to 13 C.alpha.). In addition, in H_Cin/TAB, the receptor-binding pocket is completely conserved, although involved in the knotAll residues in the complex show similar configurations in both structures (FIG. 6). This was confirmed by PISA analysis, in which H further contains 11 electrostatic bonds_ChSyt1 interaction calculation interface of/TABAnd having 7 electrostatic bonds

H of (A) to (B)_CrSyt2 interface (PDB 4KBB) was comparable. The recognition mechanism is mainly based on strong protein-protein hydrophobic interactions. Small differences in contact surface area and number of electrostatic interactions can be explained by sequence variations between hSyt1 and rSyt2, in particular for the C-terminal half of the peptide.

H_CThe third receptor contained in the/TAB-3R structure corresponds to the GD1a carbohydrate for which a clear electron density is observed from Gal2 to Sia5 (FIG. 4). For Glc1 and Sia6, no electron density was seen, as could be expected from the non-interacting flexible carbohydrate moiety. The ganglioside-receptor binding site has been studied in detail, and H complexed with GD1a_CThe crystal structure of/B has confirmed that this serotype is preferred for the terminal Sia (. alpha.2-3) Gal moiety (Bertnsson et al, 2013; Rummel, 2016). TriRecaBTox design for integration of H_Cthe/B binding pocket, and a comparison of the two structures (fig. 7) shows that the key residues of the binding pocket (S1260, W1262, Y1263) are completely conserved and interact with GD1a, according to the native toxin. H when combined with GD1a-_CMost of the binding sites remained unchanged when compared to/B, but there were few notable exceptions. At H_Cin/TAB-3R, the side chain of N1122 is turned away from the ligand, while its H_Cthe/B equivalent N1105 forms a direct hydrogen bond with Sia 5. This is compensated to some extent by the position I1257, which is identical to H_CI1240 in the GD1a Structure (where they are spaced apart)

) In contrast, in H_CThe water repellent property of the water repellent agent is stronger than that of Sia5 in the case of the water repellent agent/TAB-3RInteraction (A) with

Distance (d).

In its entirety, from H_CThe results of the/TAB-3R crystal structure confirm that a single treecabtox molecule is capable of binding to SV2 receptor, synaptotagmin receptor and its ganglioside receptor simultaneously in a manner that repeats the binding mechanism of the parent BoNT/a and/B.

Production and characterization of full-length, inactive TriRecaBTox.

By establishing H_C[ binding ability of TAB ], the inventors subsequently expressed and purified a full-length, catalytically inactive TriRecaBTox (BoNT/TAB; SEQ. ID. No. 3). To this end, the inventors designed a synthetic gene encoding 1311 amino acids and containing three BoNT domains, among which LC and H_NCorresponding to the BoNT/A domain, said functional domain being associated with H_C[ TAB ] bonding. Three mutations at the catalytic site (E224Q/R363A/Y366F) were included for safety reasons (Rossetto et al, 2001; Binz et al, 2002). According to H as described above_CTAB construct, protein sequence was codon optimized for expression in e.coli (e.coli) and cloned into pET-28a (+) with N-terminal poly-histidine tag. Detailed information is provided in the methods section. The inventors showed that BoNT/TAB can be expressed as a soluble protein of approximately 152 kDa. The initial method used for purification yielded a limited amount of heterogeneous material (FIG. 8; FIG. 13), but further purification using methods such as ion exchange or hydrophobic interaction chromatography should help to obtain a more pure material and eliminate residual host cell proteins visible by gel electrophoresis. Recently, recombinant BoNT/B constructs with a purity of more than 80% were generated using this method (Elliot et al, 2017).

Additional characterization was performed and confirmed the presence of the histidine tag, and although the response to the probe antibody was very weak compared to the control (fig. 8B), there was a discernible weak band at the correct size. The assay also showed cross-reactivity with contaminants of about 70 kDa. Furthermore, BoNT/TAB was determined to be intrinsically H resistant, as expected_CA (FIG. 8C) and H_Cthe/B (FIG. 8C) antisera reacted because it should contain epitopes from both binding domains.

Controlled activation of TriRecABTox.

BoNT/TAB with a factor Xa cleavage site, IEGR [ 442-. The full length BoNT/TAB sample (seq. id No.5) described above was used to perform the activation assay. Although the samples were heterogeneous, complete activation was achieved after incubation of BoNT/TAB with factor Xa overnight at 4 ℃ in a ratio of 1. mu.g protease to 50. mu.g toxin (FIG. 9B). Gel electrophoresis shows the separation of BoNT/TAB into two fragments of approximately 100 and 50kDa, which most likely correspond to HC and LC, respectively, when performed in the presence of a reducing agent. These two chains are held together by a disulfide bond between C430 and C458, thus accounting for a single band of approximately 150kDa under non-reducing conditions. Bands corresponding to HC and LC are also visible under non-reducing conditions and may be caused by some level of reduction of disulfide bonds during sample preparation, however these bands are not clearly visible in the unactivated control.

In general, the activation assay provides firstly evidence that the protein produced corresponds to an engineered BoNT/TAB and secondly a step in which activation can be successfully controlled to a double-stranded molecule. Thus, this step may be included in the production of active full-length treecabtox.

Optimization of BoNT/TAB

Materials and methods

Constructs. The vector encoding H was cloned by GenScript (NJ, USA) in pET28(a) as described above_CcDNA and variants of TAB. BoNT/TAB2.1.3 was cloned in the pET29(a) vector by Toxogen GmbH (Hannover, Germany).

Protein expression and purification. As above for H_CThe TAB variant.

By reaction with a compound of formula (I) and of formula (II)_CProcedure similar to that used for/TAB (affinity chromatography and gel filtration), BoNT/TAB2.1.3 was generated by Toxogen GmbH (Hannover, Germany). In addition, the passing concentration isActivation and tag removal of BoNT/TAB2.1.3 was performed with 0.05U/. mu.g of thrombin, and BoNT/TAB2.1.3 was further purified by gel filtration. Samples were stored in 25mM HEPES pH7.2 with 200mM NaCl and 5% glycerol.

And (4) protein characterization. As described above (gel electrophoresis using NuPAGE 4-12% Bis-Tris gel).

X-ray crystallography. By mixing 6.5mg/ml of H at room temperature_C/TAB2.1 with 1mg/ml SV2C-L4 (recombinant human SV2C extracellular loop-4 [ residue 475-]) Samples for crystallization were prepared by preincubation for 15min with 1mM hSyt1 peptide (GEGKEDAFSKLKEKFMNELHK, synthesized by GenScript, USA) and 4mM GD1a oligosaccharide (Elicityl, France). Using a dropping device, crystals were grown by mixing with 100nl of a 200nl sample of stock solution consisting of 20% v/v polyethylene glycol 3350, 0.2M potassium citrate (JCSG-plus screen B12, Molecular Dimensions, United Kingdom) and incubated at 21 ℃. Crystals appeared within 1 week and were transferred to a cryo-ring and frozen in liquid nitrogen. Diffraction data was collected at workstation 104-1 equipped with a Diamond light source (Didcot, UK) with a PILATUS-6M detector (Dectiris, Switzerland). At 100 ℃ K, collect from the single crystalThe complete data set of (2). The raw data images were processed and scaled by DIALS (Gildea et al 2014) and AIMLESS (Evans 2006) using CCP4 suite 7.0(CCP4, 1994).

By H previously determined in PHASER_CStructure of/TAB is subject to molecular replacement (McCoy et al, 2007). The working model was refined using REFMAC5(Murshudov et al, 2011) and manually adjusted by COOT (Emsley et al, 2010). Water molecules are added at positions where the Fo-Fc electron density peak is greater than 3 σ, and potential hydrogen bonds may be added. Verification was performed by MOLPROBITY (Chen et al, 2010).

Ramachandran statistics showed that 97.0% of all residues were in the most favorable region, with only one outlier in the unacceptable region. The crystallography statistics are summarized in table X1.

_{C C}Optimized generation of H/TAB, H/TAB2.1

Analysis of H binding to its three receptors_CThe crystal structure of/TAB to identify potential sites that can be modified to improve the stability and function of the molecule.

In particular, analysis of local temperature factors (B-factors) within the crystal structure, where a high B-factor indicates regional disorder, may be understood as an indication of local stability of the protein. From this analysis, it is believed that H consists of residues D357 to N362(seq. id No.6)_CThe ring at the interface between the two subfields of/TAB (labeled "ring 360") is optimized (see FIG. 14). Residues G360 and N362(SEQ ID. No.1) were modified to their equivalent residues in BoNT/B and mutated to P360 and Y362, respectively, to introduce the marker H_CThe sequence of the novel construct of/TAB 2.1(SEQ ID No. 6).

Plasmids of this new construct were prepared by site-directed mutagenesis (GenScript, USA) and used for H_C[ TAB2.1 in Escherichia coli (E.coli) recombinant expression. Protocol used and for H_CThe same was used for the production of/TAB (see original methods section for expression and purification). We show that H can be expressed and partially purified using affinity chromatography and size exclusion techniques_C[ FIG. 15 ] TAB2.1. The sample presents some low molecular weight contaminants that may correspond to residual host cell proteins. Other purification steps using methods such as ion exchange or hydrophobic interaction chromatography should help to obtain high purity samples.

In the domain [ residue 475-565 ] of human SV2C lumen]Human Syt1 peptide [ residues 34-53 ]]And GD1a carbohydrate in the crystallization test used purified H_C[ SEQ ID No.6 ] of TAB2.1. High resolution diffraction is obtainedAnd a complete data set can be collected (table 2). Using H bound to its three receptors_CCrystal structure of/TAB (H)_C[ TAB-3R) ], the structure was analyzed by molecular replacement. The new structure shows all elements in H_CThe ones visible in/TAB-3R and provided according to H_C/TAB，H_CExperimental evidence that/TAB 2.1 can bind to three receptors simultaneously. B-factorShows improved stability for ring "360" (D357 to Y362; FIG. 14). Overall, H_CThe behavior of/TAB 2.1 is similar to HC/TAB, with both constructs showing comparable profiles in terms of yield and purity.

Table x1. x-ray crystallography: data collection and refinement statistics

The values in parentheses are the values of the highest resolution shell.

_CProduction of more soluble variant H/TAB2.1.3

To prepare for H_CFuture functional analysis of the TAB variants, let H_Cthe/TAB 2.1 is suitable for compatibility with the recently described sortase ligation experiments (Zhang et al, 2017). This experiment allows for a safe and controlled reconstitution of full-length, active bonts that can be used to test activity. This construct corresponds to an N-terminally truncated H with a cleavable N-terminal His-tag_C[ TAB2.1 ] and this is marked as H_C/TAB2.1.1(SEQ. ID. No. 8). Preparation of H_C/TAB2.1.1 (GenScript) and used for expression and purification as described above (FIG. 16). We show that H can be expressed and partially purified using affinity chromatography and size exclusion techniques_Cand/TAB2.1.1. The sample presents some low molecular weight contaminants that may correspond to residual host cell proteins. Other purification steps using methods such as ion exchange or hydrophobic interaction chromatography should help to obtain high purity samples.

H_CFurther analysis of the structural characteristics of/TAB 2.1 highlights the presence of surface exposed hydrophobic loops protruding from the rest of the protein (residue 389-393, SEQ. ID. No. 6; FIG. 14 d). In addition, this loop has recently been identified as a lipid-binding element in BoNT/B and other serotypes (Stern et al, 2018). We hypothesize that this hydrophobic region may interfere with H_C/TAB solubility, a new construct was therefore designed in which the loop is truncated and replaced with a bis-asparagine motif to increase solubility. Labeling the construct as H_C/TAB2.1.3(SEQ.Id.no. 10). Preparation of H_C/TAB2.1.3 (GenScript) and used for expression and purification as described above (FIG. 16). We show that H can be expressed and partially purified using affinity chromatography and size exclusion techniques_Cand/TAB2.1.3. The sample presents some low molecular weight contaminants that may correspond to residual host cell proteins. Other purification steps using methods such as ion exchange or hydrophobic interaction chromatography should help to obtain high purity samples. Notably, with H_CComparison of TAB2.1.1 with H_C/TAB2.1.3 showed better expression yield and solubility (FIG. 16).

Production of full-Length, active BoNT/TAB2.1.3

To prepare future functional analyses of BoNT/TAB, H-based generation was performed_CThe full-length, active variant of the/TAB2.1.3 construct was labeled BoNT/TAB2.1.3(SEQ. ID. No. 12). In licensed institutions, all production steps were carried out under contractual agreements at Toxogen GmbH (Hannover, Germany). BoNT/TAB2.1.3 was cloned in pET29(a) vector and it contained a cleavable C-terminal Strep tag and poly-histidine tag, as well as an engineered thrombin cleavage site for product activation between the HC and LC domains as described above (seq. id No. 13). BoNT/TAB2.1.3 can be expressed as a soluble protein, purified and activated by thrombin (FIG. 17). The method for purification includes affinity chromatography and gel filtration, and it results in>BoNT/TAB2.1.3 product of 90% purity.

Future experiments

Receptor binding assays

Assays will be performed in which the receptor-binding properties of BoNT/TAB will be compared to BoNT/A and/or BoNT/B.

For example, a ganglioside receptor-binding assay modified according to the methods previously described will be performed. Briefly, in this ELISA, the ganglioside receptor of interest (GT1b, GD1b, GD1a, or GM1a) is immobilized in a 96-well microplate (Chen et al, 2008; Willjes et al, 2013), and then the toxins (or their binding domains) are applied and the bound material is detected with a monoclonal anti-poly-histidine antibody conjugated to horseradish peroxidase (HRP). This qualitative approach should provide sufficient information to confirm that the ganglioside receptor-binding characteristics of BoNT/TAB are similar to those of BoNT/B.

Ganglioside receptor binding ELISA. The gangliosides GT1b, GD1b, GD1a and GM1a were purchased from Carbosynth (Compton, UK). Diluting the ganglioside in methanol to reach a final concentration of 2.5 μ g/ml; 100 μ L (0.25 μ g) was applied to each well in a 96-well PVC assay plate. After evaporation of the solvent at 21 deg.C (overnight), the wells were washed (3X) with 200. mu.L PBS/0.1% (w/v) BSA. Non-specific binding sites were blocked by incubation for 2h at 21 ℃ in 200. mu.L PBS/2% (w/v) BSA. Binding assays were performed at 4 ℃ for 2h in 100. mu.L PBS/0.1% (w/v) BSA/well containing samples (serial 3-fold dilutions ranging from 6. mu.M to 0.003. mu.M). After incubation, wells were washed 3X with PBS/0.1% (w/v) BSA and then incubated with HRP-anti-His antibody (ThermoFisher # MA1-80218) (100. mu.l/well) diluted 1:2000 at 4 ℃ for 1 h. Finally, after three washing steps with PBS/0.1% (w/v) BSA, bound samples were detected using Ultra TMB (100. mu.L/well). After incubation at 21 ℃ for 5min, the reaction was terminated by adding 100. mu.L of 1M sulfuric acid. Using Tecan Infinite 200(Switzerland) measured the absorbance at 450 nm. The results were analyzed by Prism (GraphPad, La Jolla, Calif., USA) using nonlinear binding fitting.

To evaluate the binding properties to the synaptic binding protein receptor, Isothermal Titration Calorimetry (ITC) was performed in analogy to the assay described by Berntsson et al (2013). Binding of the hSyt peptide to the toxin will be measured and should provide an affinity value (K) confirming that BoNT/TAB can bind to the receptor similar to BoNT/B_d)。

Isothermal titration calorimetry. Samples were prepared by other size exclusion chromatography steps (Superdex200, GE Healthcare, Sweden) in 20mM potassium phosphate pH 7.0, 0.15M NaCl. Binding of the Syt peptide to bonts or their binding domains was measured at 25 ℃ and 750rpm on ITC200(GE Healthcare, Sweden). 200 μ L of protein solution (20 μ M) was added to the cuvette. Binding was measured by 16 stepwise injections of 2.5 μ L of added peptide (GenScript, USA) per injection at a concentration of 200 μ M. The first titration was set to 0.5 μ L and subsequently deleted in the data analysis. Data were analyzed by Origin software provided by the manufacturer

Binding to SV2C was evaluated using a Pull-down assay (Pull-down assay) as described by Benoit et al (2014). Briefly, the tagged toxin and non-tagged receptor (or vice versa) will be incubated together and loaded onto Ni-agarose, then washed and eluted. The results were shown by SDS-PAGE.

Posterior toe abduction score (DAS) determination

The efficacy of BoNT formulations can be evaluated using a mouse hind toe abduction score (DAS) assay (Broide et al, 2013). This assay measures in vivo the local muscular-weakening efficacy of the toxin following intramuscular injection into the skeletal muscle of the lower limb of a mouse or rat. The toxin causes a measurable dose-dependent decrease in the animal's ability to produce a characteristic lower limb startle response. This non-lethal approach has been commonly used to estimate the pharmacological properties of different BoNT serotypes or derivatives, such as the recently described recombinant BoNT/B molecules (Elliot et al, 2017). Similar methods will be used to evaluate the efficacy and duration of action of BoNT/TAB compared to BoNT/A or/B.

Discussion of the related Art

In this study, the inventors describe how to use the structural and molecular details of the binding mechanism of BoNT/a and/B to engineer a new molecule, treecabtox, with improved cell recognition capabilities. Rigorous multi-scale comparisons of BoNT/a and/B structures enabled the present inventors to identify key elements required to retain the complete toxin scaffold in a single molecule on which the receptor binding sites of SV2, synaptotagmin and gangliosides are integrated. The newly created design includes an alternation of BoNT/a and/B elements and is optimized by protecting adaptive mutations or deletions to compensate for the newly created non-native intramolecular interface. These modifications are considered necessary to ensure that the engineered toxin BoNT/TAB can be produced as a soluble protein with the correct structure and desired activity.

The inventors first evaluated the molecular structure of the compound by_CStability of the design to generate the binding Domain on TAB itself, said H_C/TAB through inColi (e.coli) with modified receptor recognition function. As a soluble protein that can be partially purified, H having an N-terminal poly-histidine tag was expressed_CTAB, thus indicating the viability of the engineered construct. In a second step, the inventors proceeded with the generation of full-length BoNT/TAB constructs in catalytically inactive form. Again, the inventors showed that it could be expressed as a 153kDa soluble protein and partially purified by standard liquid chromatography techniques. H_CThe presence of poly-histidine tags on both/TAB and BoNT/TAB enables purification by affinity chromatography on a Ni-agarose matrix. Other affinity methods can be used and include affinity tags that should preferentially be located on the N-terminus of the protein to avoid interfering with receptor binding. Although the initial formulation showed a non-uniform sample purity, optimization of the purification process should result in a product that is drug-standard. The active form that should be supplemented with BoNT/TAB will have similar overall structure and binding properties as the inactive molecules used in this study. The present inventors have entered into an agreement with Toxogen GmbH (Hannover, Germany) to produce an active form of BoNT/TAB (BoNT/TAB2.1.3) that is successfully purified by a removable C-terminal tag so as not to interfere with receptor binding.

In addition, post-translational cleavage of single-chain BoNTs into di-chain molecules is an essential step in toxin activity (DasGupta and Sathyamoorthy, 1985; Shone et al, 1985). Although native toxins are typically activated by host proteases, any recombinant BoNT product requires processing by an exopeptidase. Early work on toxins showed that trypsin could non-specifically cleave BoNT/A into the active di-chain form (Shone et al, 1985), however this could lead to undesired additional degradation of the toxin. Recently, recombinant technology has enabled the engineering of specific protease recognition motifs within proteins of interest, thus providing better control over BoNT activation strategies (Sutton et al, 2005). Herein, the inventors have included a factor Xa site between LC and HC and observed complete activation of the toxin, thus demonstrating the effectiveness of the enzyme. Future BoNT/TAB production should introduce a purification platform that enables activation of the toxin and then removal of the exoprotease from the final product. Although factor Xa appears to be adequate, other enzymes can be tested and proved successful in achieving acceptable activation yields. The present inventors have agreed with Toxogen GmbH (Hannover, Germany) to produce an active form (BoNT/TAB2.1.3) that is successfully activated by exothrombin and purified to homogeneity for BoNT/TAB.

As verification H_CStructural integrity of/TAB and means to confirm its improved functionality, the inventors co-crystallized purified samples complexed with human SV2C, human Syt1, and GD1a carbohydrates. High resolutionResolving the X-ray crystal structure of the complex and providing a single H_CConclusive experimental evidence that the/TAB molecule can bind to all three receptors simultaneously. In addition, with H having its respective receptor_CA and H_CComparison of the known structures of/B shows H_Cthe/TAB follows almost the same binding mechanism.

Although the crystal structure indicates H_Cthe/TAB can at least meet its purpose in vitro, but other biochemical experiments need to be performed to fully characterize its receptor binding properties. These would include pull-down assays and ITC assays using protein receptors and ganglioside receptor binding ELISA. BoNT/TAB is expected to be similar to BoNT/A for SV2 receptor binding and to BoNT/B for ganglioside receptor and synaptotagmin receptor binding. In addition, in vivo experiments will provide a major indication of the true potential of BoNT/TAB as a therapeutic. The mouse DAS assay has been classically used to evaluate BoNT formulations (Broide et al, 2013) and should enable the inventors to determine the efficacy and duration of action of our molecule compared to the off-the-shelf product.

In addition, the design of BoNT/TAB can be further optimized by modifying some of the sequence elements to improve its biochemical properties and stability. These changes may include deletions or mutations that result in soluble bonts that are still capable of binding to three receptors simultaneously. The inventors succeeded in producing more stable variants (H)_CTAB2.1) and produces more soluble variants (H) in higher yields_C/TAB2.1.3)。

It should be added that from a safety point of view, BoNT/TAB does not represent a new threat, since it originates from two existing serotypes. It is expected to be recognized by currently available antitoxins, such as the botulinum antitoxin heptavalent BAT or other approved antidotes for BoNT/a and/B.

Serological types a and B are the only approved marketed bonts. Although BoNT/a is the main therapeutically used toxin, molecules with low immunogenicity and high potency will provide a safer alternative (Naumann et al, 2013). There have been multiple attempts to improve the properties of BoNT to increase its pharmacological potential (Masuyer et al, 2014). Examples of recent success include the study by Tao et al (2017), where mutations engineered at key positions of BoNT/B (E1191M/S1199Y) provide toxins with higher affinity for human synaptotagmin 2 receptors and show about 11-fold higher potency in blocking neurotransmission compared to the wild type. Elliott et al (2017) took another approach to improve the efficacy of BoNT, where they analyzed the effect of a single mutation (S201P) known to increase the catalytic activity of BoNT/B' S substrate. In this case, the BoNT/B mutant does not show any advantage over the wild type in the multicellular-based assay and in vivo. Overall, these two studies with BoNT/B indicate that the limiting step in toxin potency is in initial neuronal recognition, rather than subsequent intracellular activity.

Earlier studies aimed at combining the binding properties of one serotype with the catalytic activity of another led to the design of chimeric molecules in which the entire domain was exchanged (Wang et al, 2008,2012; Rummel et al, 2011). More specifically, Rummel et al (2011) and Wang et al (2012) designed and tested H by BoNT/A_N+ LC Domain bound H_CSimilar molecules consisting of/B domain. These recombinant toxins are reported to show increased potency and induce longer effects in mice compared to wild-type BoNT/a (Kutschenko et al, 2017). When evaluating the C-terminal subdomain (H) of BoNT/B bound by the complementary domain of serogroup A_CC) (i.e. LC + H)_N+H_Cn) Similar observations were obtained for constructs composed and showing 4-fold higher potency than wild-type (Rummel et al, 2011). All of the aboveThe molecules share the following facts: they will recognize only two receptors in BoNT/B, synaptotagmin and gangliosides. These results indicate that prolonged action and higher efficacy can be achieved due to the greater LC/a access allowed by the more prevalent BoNT/B receptors on neurons. In addition, these chimeric molecules do not take into account possible intra-domain intramolecular conflicts that may arise from the combination of domains from different serotypes and that may affect the potential of these products.

Given the recent results of studies on BoNT engineering, it is clear that modifying initial cell recognition is one of the most effective ways to improve the pharmacological properties of therapeutic products. Thus, BoNT/TAB, a single product that was successfully engineered to recognize the SV2 receptor as well as the BoNT/B receptor, synaptotagmin, and ganglioside, showed great potential and could be more potent than wild-type BoNT/a and/B.

The main innovation of BoNT/TAB is the design of binding domains that allow multi-receptor interaction. Current evidence indicates H_CThe translocation of/TAB to BoNT/A and the binding of the catalytic domain should provide the most potent molecule (as designed in BoNT/TAB). However, when combined with functional domains of other serotypes, H_Cthe/TAB may still be of interest (FIG. 10 a). In addition, H_Cthe/TAB can also be combined with other proteins of interest used as pharmacological tools (fig. 10b) to study synaptic processes. The in vivo assay performed by BoNT/TAB should demonstrate its utility for this purpose.

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Sequence listing

<110> Parr, Stelmak

<120>TricepTox

<130>P41705056PCT00

<160>14

<170>PatentIn version 3.5

<210>1

<211>437

<212>PRT

<213> Artificial sequence

<220>

<223>Hc/TriRecABTox

<400>1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr Gly Glu

100 105 110

Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys Gln Arg Val Val

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Lys Asp Ser Pro Val Gly Glu Ile Leu Gly Pro Arg Gly Ser Val Met

260 265 270

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

275 280 285

Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp Asp Ile Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn

405 410 415

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

420 425 430

Gly Glu Arg Pro Leu

435

<210>2

<211>1314

<212>DNA

<213> Artificial sequence

<220>

<223> codon optimized Hc/TriRecaBTox for expression in E.coli (E. coli)

<400>2

aagaacatta tcaacaccag catcctgaac ctgcgctacg agagcaacca cctgatcgac 60

ctgagccgct acgcgagcaa gattaacatc ggtagcaagg tgaactttga cccgattgat 120

aaaaaccaga tccaactgtt caacctggaa agcagcaaga tcgaagtgat tctgaaaaac 180

gcgattgttt ataacagcat gtacgaaaac ttcagcacca gcttttggat ccgtattccg 240

aagtatttta acagcatcag cctgaacaac gaatacacca tcattaactg catggagaac 300

aacagcggtt ggaaagtgag cctgaactac ggcgaaatca tttggaccct gcaggacacc 360

caagagatca agcagcgtgt ggttttcaag tacagccaaa tgatcaacat cagcgattac 420

atcaaccgtt ggattttcgt taccatcacc aacaaccgtc tgaacaacag caagatctac 480

attaacggtc gtctgattga ccagaaaccg atcagcaacc tgggcaacat tcacgcgagc 540

aacaacatca tgttcaagct ggacggttgc cgtgataccc accgttatat ctggattaag 600

tacttcaacc tgtttgataa agagctgaac gaaaaggaga ttaaagacct gtatgataac 660

cagagcaaca gcggtatcct gaaggacttt tggggcgatt atctgcaata cgacaaaccg 720

tactatatgt tcaacgcggg taacaagaac agctacatta aactgaagaa agatagcccg 780

gtgggtgaaa tcctgggtcc gcgtggcagc gttatgacca ccaacatcta tctgaacagc 840

agcctgtacc gtggcgagaa gttcatcatt cgtcgtaaaa gcaacagcca gagcattaac 900

gacgatatcg tgcgtaacga agactacatt tatctggatt tctttaacct gaaccaagag 960

tggcgtgttt acacctacaa gtacttcaag aaagaggaag agaagctgtt cctggcgccg 1020

atcagcgaca gcgatgaatt ctacaacacc atccaaatca aggaatacga cgagcagggt 1080

accaacagct gccaactgct gttcaagaaa gacgaagaga gcaccgatga aatcggtctg 1140

atcggcattc accgtttcta cgagagcggc atcgtgttcg aagagtacaa ggattacttc 1200

tgcatcagca agtggtatct gaaagaggtt aagcgtaaac cgtacaacct gaaactgggc 1260

tgcaactggc aatttattcc ggtggatgat ggctggggtg aacgtccgct gtaa 1314

<210>3

<211>1311

<212>PRT

<213> Artificial sequence

<220>

<223> full-length inactive TriRecaBTox with engineered activation sites

<400>3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

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 Ala Lys Thr Phe 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 Ile Glu Gly Arg Asp Lys

435 440 445

Gly Tyr Asn Lys Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile

515 520 525

Ile Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

Thr Leu Ile Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Gly Thr Asn Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275

Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu

1280 1285 1290

Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Asp Gly Trp Gly Glu

1295 1300 1305

Arg Pro Leu

1310

<210>4

<211>3936

<212>DNA

<213> Artificial sequence

<220>

<223> full-length inactive TriRecaBTox with engineered activation sites,

sequences optimized for expression of codons in E.coli (E. coli)

<400>4

atgccgttcg tgaataagca gttcaactac aaagatccgg ttaatggcgt ggacatcgcg 60

tacatcaaaa tcccgaatgc gggtcagatg cagccggtga aggcgttcaa aatccacaac 120

aaaatttggg ttatcccgga gcgtgacacc tttaccaacc cggaggaagg tgatctgaac 180

ccgccgccgg aagcgaaaca agtgccggtt agctactatg acagcaccta tctgagcacc 240

gacaacgaga aggataacta cctgaagggc gtgaccaaac tgttcgaacg tatctacagc 300

accgatctgg gtcgtatgct gctgaccagc attgttcgtg gcatcccgtt ttggggtggc 360

agcaccatcg acaccgaact gaaagtgatt gataccaact gcattaacgt tatccagccg 420

gatggtagct accgtagcga ggaactgaac ctggtgatca ttggcccgag cgcggacatc 480

attcagtttg agtgcaagag cttcggtcac gaagttctga acctgacccg taacggttac 540

ggcagcaccc aatatatccg tttcagcccg gatttcacct ttggcttcga ggaaagcctg 600

gaagtggaca ccaacccgct gctgggtgcg ggcaagtttg cgaccgaccc ggcggttacc 660

ctggcgcacc agctgatcca tgcgggtcac cgtctgtacg gcattgcgat caacccgaac 720

cgtgtgttca aagttaacac caacgcgtac tatgagatga gcggtctgga agtgagcttt 780

gaggaactgc gtaccttcgg tggccacgac gcgaagttta tcgatagcct gcaggagaac 840

gaattccgtc tgtactacta caacaagttc aaggacatcg cgagcaccct gaacaaggcg 900

aaaagcattg tgggtaccac cgcgagcctg caatacatga agaacgtttt caaggagaag 960

tacctgctga gcgaagatac cagcggcaag tttagcgtgg acaagctgaa attcgataag 1020

ctgtataaaa tgctgaccga gatctacacc gaagataact tcgtgaagtt ctttaaagtt 1080

ctgaacgcga aaacctttct gaacttcgac aaggcggttt ttaaaattaa catcgtgccg 1140

aaggttaact acaccatcta tgatggtttc aacctgcgta acaccaacct ggcggcgaac 1200

tttaacggcc agaacaccga gattaacaac atgaacttta ccaagctgaa aaacttcacc 1260

ggtctgtttg aattctataa actgctgtgc gtgcgtggca tcattaccag caagaccaaa 1320

agcctgatcg aaggtcgtga caagggctac aacaaagcgc tgaacgatct gtgcattaaa 1380

gttaacaact gggacctgtt ctttagcccg agcgaggaca acttcaccaa cgatctgaac 1440

aagggcgagg aaatcaccag cgacaccaac attgaagcgg cggaggaaaa catcagcctg 1500

gatctgattc agcaatatta cctgaccttt aacttcgaca acgagccgga aaacattagc 1560

atcgagaacc tgagcagcga catcattggt cagctggagc tgatgccgaa catcgaacgt 1620

ttcccgaacg gcaagaaata cgaactggat aaatatacca tgttccacta cctgcgtgcg 1680

caagagtttg aacacggcaa gagccgtatt gcgctgacca acagcgtgaa cgaggcgctg 1740

ctgaacccga gccgtgttta taccttcttt agcagcgact acgtgaagaa agttaacaaa 1800

gcgaccgagg cggcgatgtt cctgggttgg gtggaacagc tggtttacga ctttaccgat 1860

gaaaccagcg aggtgagcac caccgacaaa attgcggata tcaccatcat tatcccgtat 1920

atcggtccgg cgctgaacat tggcaacatg ctgtacaagg acgattttgt gggtgcgctg 1980

atcttcagcg gcgcggttat cctgctggag ttcattccgg aaattgcgat cccggtgctg 2040

ggtacctttg cgctggttag ctacatcgcg aacaaggtgc tgaccgttca aaccattgat 2100

aacgcgctga gcaagcgtaa cgagaaatgg gacgaagtgt ataaatacat cgttaccaac 2160

tggctggcga aggttaacac ccagattgac ctgatccgta agaaaatgaa agaggcgctg 2220

gaaaaccaag cggaggcgac caaggcgatt atcaactatc agtacaacca atacaccgag 2280

gaagagaaaa acaacattaa cttcaacatc gacgatctga gcagcaagct gaacgaaagc 2340

atcaacaaag cgatgattaa catcaacaag tttctgaacc agtgcagcgt gagctatctg 2400

atgaacagca tgattccgta cggtgttaag cgtctggagg acttcgatgc gagcctgaag 2460

gacgcgctgc tgaaatatat ctacgataac cgtggtaccc tgattggcca agtggaccgt 2520

ctgaaggata aagttaacaa caccctgagc accgatatcc cgttccagct gagcaaatat 2580

gtggacaacc aacgtctgct gagcaccttt accgagtaca tcaagaacat tatcaacacc 2640

agcattctga acctgcgtta tgaaagcaac cacctgatcg acctgagccg ttacgcgagc 2700

aagattaaca tcggtagcaa agttaacttc gacccgatcg ataaaaacca gattcaactg 2760

tttaacctgg agagcagcaa gattgaagtg atcctgaaaa acgcgatcgt ttacaacagc 2820

atgtatgaga actttagcac cagcttctgg attcgtatcc cgaaatattt caacagcatt 2880

agcctgaaca acgagtacac cattatcaac tgcatggaaa acaacagcgg ttggaaggtg 2940

agcctgaact acggcgagat tatctggacc ctgcaggaca cccaagaaat caagcagcgt 3000

gtggttttca agtacagcca aatgatcaac atcagcgatt acattaaccg ttggatcttt 3060

gttaccatta ccaacaaccg tctgaacaac agcaaaattt acatcaacgg tcgtctgatc 3120

gaccagaagc cgattagcaa cctgggcaac atccacgcga gcaacaacat tatgttcaag 3180

ctggacggtt gccgtgatac ccaccgttat atttggatca agtacttcaa cctgttcgat 3240

aaggagctga acgagaagga aatcaaagac ctgtatgata accagagcaa cagcggtatt 3300

ctgaaagact tctggggcga ttacctgcaa tatgacaagc cgtattacat gtttaacgcg 3360

ggtaacaaga acagctacat caaactgaag aaagatagcc cggtgggtga aattctgggt 3420

ccgcgtggca gcgttatgac caccaacatc tatctgaaca gcagcctgta ccgtggcgaa 3480

aagttcatta tccgtcgtaa aagcaacagc cagagcatca acgacgatat tgtgcgtaac 3540

gaggactata tctacctgga tttctttaac ctgaaccaag aatggcgtgt ttacacctac 3600

aagtacttca agaaagaaga ggaaaagctg tttctggcgc cgattagcga cagcgatgaa 3660

ttctataaca ccattcagat caaagagtac gacgaacagg gtaccaacag ctgccaactg 3720

ctgtttaaga aagacgagga aagcaccgat gagatcggtc tgattggcat ccaccgtttt 3780

tacgaaagcg gcatcgtgtt cgaggaatac aaggattact tctgcatcag caagtggtat 3840

ctgaaagagg ttaagcgtaa accgtacaac ctgaaactgg gctgcaactg gcaatttatt 3900

ccggtggatg atggctgggg tgaacgtccg ctgtaa 3936

<210>5

<211>1311

<212>PRT

<213> Artificial sequence

<220>

<223> full-length inactive TreRecABTox with engineered activation sites

<400>5

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 Ile Glu Gly Arg Asp Lys

435 440 445

Gly Tyr Asn Lys Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile

515 520 525

Ile Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

Thr Leu Ile Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser

965 970 975

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

980 985 990

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

9951000 1005

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

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

Gly Glu Lys Phe Ile Ile Arg Arg LysSer Asn Ser Gln Ser Ile

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Gly Thr Asn Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275

Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu

1280 1285 1290

Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Asp Gly Trp Gly Glu

1295 1300 1305

Arg Pro Leu

1310

<210>6

<211>437

<212>PRT

<213> Artificial sequence

<220>

<223> Hc/TAB2.1, optimized Ring 360

<400>6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr Gly Glu

100 105 110

Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys Gln Arg Val Val

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Lys Asp Ser Pro Val Gly Glu Ile Leu Gly Pro Arg Gly Ser Val Met

260 265 270

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

275 280 285

Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp Asp Ile Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn

405 410 415

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

420 425 430

Gly Glu Arg Pro Leu

435

<210>7

<211>1314

<212>DNA

<213> Artificial sequence

<220>

<223> Hc/TAB2.1, codon optimization for expression in E.coli (E. coli)

<400>7

aagaacatta tcaacaccag catcctgaac ctgcgctacg agagcaacca cctgatcgac 60

ctgagccgct acgcgagcaa gattaacatc ggtagcaagg tgaactttga cccgattgat 120

aaaaaccaga tccaactgtt caacctggaa agcagcaaga tcgaagtgat tctgaaaaac 180

gcgattgttt ataacagcat gtacgaaaac ttcagcacca gcttttggat ccgtattccg 240

aagtatttta acagcatcag cctgaacaac gaatacacca tcattaactg catggagaac 300

aacagcggtt ggaaagtgag cctgaactac ggcgaaatca tttggaccct gcaggacacc 360

caagagatca agcagcgtgt ggttttcaag tacagccaaa tgatcaacat cagcgattac 420

atcaaccgtt ggattttcgt taccatcacc aacaaccgtc tgaacaacag caagatctac 480

attaacggtc gtctgattga ccagaaaccg atcagcaacc tgggcaacat tcacgcgagc 540

aacaacatca tgttcaagct ggacggttgc cgtgataccc accgttatat ctggattaag 600

tacttcaacc tgtttgataa agagctgaac gaaaaggaga ttaaagacct gtatgataac 660

cagagcaaca gcggtatcct gaaggacttt tggggcgatt atctgcaata cgacaaaccg 720

tactatatgt tcaacgcggg taacaagaac agctacatta aactgaagaa agatagcccg 780

gtgggtgaaa tcctgggtcc gcgtggcagc gttatgacca ccaacatcta tctgaacagc 840

agcctgtacc gtggcgagaa gttcatcatt cgtcgtaaaa gcaacagcca gagcattaac 900

gacgatatcg tgcgtaacga agactacatt tatctggatt tctttaacct gaaccaagag 960

tggcgtgttt acacctacaa gtacttcaag aaagaggaag agaagctgtt cctggcgccg 1020

atcagcgaca gcgatgaatt ctacaacacc atccaaatca aggaatacga cgagcagccg 1080

acctatagct gccaactgct gttcaagaaa gacgaagaga gcaccgatga aatcggtctg 1140

atcggcattc accgtttcta cgagagcggc atcgtgttcg aagagtacaa ggattacttc 1200

tgcatcagca agtggtatct gaaagaggtt aagcgtaaac cgtacaacct gaaactgggc 1260

tgcaactggc aatttattcc ggtggatgat ggctggggtg aacgtccgct gtaa 1314

<210>8

<211>432

<212>PRT

<213> Artificial sequence

<220>

<223>Hc/TAB2.1.1

<400>8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Ile Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn

85 90 95

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

100 105 110

Gln Asp Thr Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gly Glu Ile Leu Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Tyr Lys Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

<210>9

<211>1299

<212>DNA

<213> Artificial sequence

<220>

<223> Hc/TAB2.1.1, codon optimized for expression in E.coli (E. coli)

<400>9

accagcatcc tgaacctgcg ctacgagagc aaccacctga tcgacctgag ccgctacgcg 60

agcaagatta acatcggtag caaggtgaac tttgacccga ttgataaaaa ccagatccaa 120

ctgttcaacc tggaaagcag caagatcgaa gtgattctga aaaacgcgat tgtttataac 180

agcatgtacg aaaacttcag caccagcttt tggatccgta ttccgaagta ttttaacagc 240

atcagcctga acaacgaata caccatcatt aactgcatgg agaacaacag cggttggaaa 300

gtgagcctga actacggcga aatcatttgg accctgcagg acacccaaga gatcaagcag 360

cgtgtggttt tcaagtacag ccaaatgatc aacatcagcg attacatcaa ccgttggatt 420

ttcgttacca tcaccaacaa ccgtctgaac aacagcaaga tctacattaa cggtcgtctg 480

attgaccaga aaccgatcag caacctgggc aacattcacg cgagcaacaa catcatgttc 540

aagctggacg gttgccgtga tacccaccgt tatatctgga ttaagtactt caacctgttt 600

gataaagagc tgaacgaaaa ggagattaaa gacctgtatg ataaccagag caacagcggt 660

atcctgaagg acttttgggg cgattatctg caatacgaca aaccgtacta tatgttcaac 720

gcgggtaaca agaacagcta cattaaactg aagaaagata gcccggtggg tgaaatcctg 780

ggtccgcgtg gcagcgttat gaccaccaac atctatctga acagcagcct gtaccgtggc 840

gagaagttca tcattcgtcg taaaagcaac agccagagca ttaacgacga tatcgtgcgt 900

aacgaagact acatttatct ggatttcttt aacctgaacc aagagtggcg tgtttacacc 960

tacaagtact tcaagaaaga ggaagagaag ctgttcctgg cgccgatcag cgacagcgat 1020

gaattctaca acaccatcca aatcaaggaa tacgacgagc agccgaccta tagctgccaa 1080

ctgctgttca agaaagacga agagagcacc gatgaaatcg gtctgatcgg cattcaccgt 1140

ttctacgaga gcggcatcgt gttcgaagag tacaaggatt acttctgcat cagcaagtgg 1200

tatctgaaag aggttaagcg taaaccgtac aacctgaaac tgggctgcaa ctggcaattt 1260

attccggtgg atgatggctg gggtgaacgt ccgctgtaa 1299

<210>10

<211>424

<212>PRT

<213> Artificial sequence

<220>

<223> Hc/TAB2.1.3, improved solubility

<400>10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Ile Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn

85 90 95

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

100 105 110

Gln Asp Thr Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gly Glu Ile Leu Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Tyr Lys Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Asp Gly Trp Gly Glu Arg Pro Leu

420

<210>11

<211>1275

<212>DNA

<213> Artificial sequence

<220>

<223> Hc/TAB2.1.3, codon optimized for expression in E.coli (E. coli)

<400>11

accagcatcc tgaacctgcg ctacgagagc aaccacctga tcgacctgag ccgctacgcg 60

agcaagatta acatcggtag caaggtgaac tttgacccga ttgataaaaa ccagatccaa 120

ctgttcaacc tggaaagcag caagatcgaa gtgattctga aaaacgcgat tgtttataac 180

agcatgtacg aaaacttcag caccagcttt tggatccgta ttccgaagta ttttaacagc 240

atcagcctga acaacgaata caccatcatt aactgcatgg agaacaacag cggttggaaa 300

gtgagcctga actacggcga aatcatttgg accctgcagg acacccaaga gatcaagcag 360

cgtgtggttt tcaagtacag ccaaatgatc aacatcagcg attacatcaa ccgttggatt 420

ttcgttacca tcaccaacaa ccgtctgaac aacagcaaga tctacattaa cggtcgtctg 480

attgaccaga aaccgatcag caacctgggc aacattcacg cgagcaacaa catcatgttc 540

aagctggacg gttgccgtga tacccaccgt tatatctgga ttaagtactt caacctgttt 600

gataaagagc tgaacgaaaa ggagattaaa gacctgtatg ataaccagag caacagcggt 660

atcctgaagg acttttgggg cgattatctg caatacgaca aaccgtacta tatgttcaac 720

gcgggtaaca agaacagcta cattaaactg aagaaagata gcccggtggg tgaaatcctg 780

ggtccgcgtg gcagcgttat gaccaccaac atctatctga acagcagcct gtaccgtggc 840

gagaagttca tcattcgtcg taaaagcaac agccagagca ttaacgacga tatcgtgcgt 900

aacgaagact acatttatct ggatttcttt aacctgaacc aagagtggcg tgtttacacc 960

tacaagtact tcaagaaaga ggaagagaag ctgttcctgg cgccgatcag cgacagcgat 1020

gaattctaca acaccatcca aatcaaggaa tacgacgagc agccgaccta tagctgccaa 1080

ctgctgttca agaaagacga agagagcacc gatgaaatcg gtctgatcgg cattcaccgt 1140

ttcaacaaca aggattactt ctgcatcagc aagtggtatc tgaaagaggt taagcgtaaa 1200

ccgtacaacc tgaaactggg ctgcaactgg caatttattc cggtggatga tggctggggt 1260

gaacgtccgc tgtaa 1275

<210>12

<211>1343

<212>PRT

<213> Artificial sequence

<220>

<223> BoNT/TAB2.1.3, full-length active protein

<400>12

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 AsnVal 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 Ala

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 AsnLys 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 Ala Lys Thr Phe 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 Ala Gly Ala Gly Lys Ser Leu Val Pro Arg

435 440 445

Gly Ser Ala Gly Ala Gly Ala Leu Asn Asp Leu CysIle Lys Val Asn

450 455 460

Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp

465 470 475 480

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

485 490 495

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

500 505 510

Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser

515 520 525

Asp Ile Ile Gly Gln Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro

530 535 540

Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu

545 550 555 560

Arg Ala Gln Glu Phe Glu His Gly Lys Ser Arg Ile Ala Leu Thr Asn

565 570 575

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

580 585 590

Ser Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met

595 600 605

Phe Leu Gly Trp Val Glu Gln Leu Val Tyr Asp Phe Thr AspGlu Thr

610 615 620

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

625 630 635 640

Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Lys Met Lys Glu Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile

740 745 750

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

755 760 765

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

770 775 780

Lys Ala Met Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser

785 790 795 800

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

805 810 815

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

820 825 830

Arg Gly Thr Leu Ile Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Ser Ile Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn

965 970 975

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

980 985 990

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

995 1000 1005

Gln Met Ile Asn Ile Ser Asp Tyr Ile Asn Arg Trp Ile Phe Val

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe Asp Lys

1070 1075 1080

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

1085 10901095

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

1100 1105 1110

Asp Lys Pro Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Glu Gln Gly Thr Asn Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275

Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu

1280 1285 1290

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

1295 1300 1305

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

1310 1315 1320

Val Asp Lys Leu Trp Ser His Pro Gln Phe Glu Lys Leu Glu His

1325 1330 1335

His His His His His

1340

<210>13

<211>4032

<212>DNA

<213> Artificial sequence

<220>

<223> BoNT/TAB2.1.3 sequence contained in pEt29(a) vector for expression in E.coli (E. coli)

<400>13

atgccatttg tgaacaagca gtttaactat aaggacccgg tgaacggtgt ggatatcgcg 60

tatatcaaaa tcccgaatgc gggccagatg caaccagtca aggcgttcaa gattcataac 120

aagatttggg ttattccgga acgtgatacc ttcaccaatc cggaagaagg cgacttaaac 180

ccgccgccag aagccaaaca agtgccggtg agctactatg atagcacgta tcttagcacc 240

gataatgaaa aagacaatta cctgaagggc gtgaccaagt tgttcgagcg catctacagt 300

accgacttag gccgcatgtt gttgacgagc atcgttcgcg gtatcccgtt ctggggcggc 360

tcgaccattg ataccgagtt gaaagtcatt gacacgaact gtatcaatgt tatccaaccg 420

gacggcagtt atcgcagcga ggagttaaat ttggtcatca tcggtccaag cgcagatatt 480

attcagttcg aatgcaagag cttcggccat gaggtcttga atttgacgcg caacggttac 540

ggcagcaccc aatacatccg ctttagcccg gatttcacct ttggcttcga ggagagcttg 600

gaggtggaca ccaacccgct gttaggtgcc ggcaaattcg caaccgaccc ggcagtgacg 660

ttggcgcacg cgttgattca tgcgggtcac cgcttatacg gtatcgcgat caatccgaat 720

cgcgtcttta aagtcaatac caacgcgtac tacgaaatga gcggcttaga ggttagcttt 780

gaagaattac gcaccttcgg tggccacgac gccaagttca tcgacagcct gcaggaaaat 840

gagttccgct tgtactatta caataaattc aaggacatcg cgagcacctt aaataaagca 900

aagagcattg tgggcaccac cgcaagcttg cagtacatga agaacgtatt taaggaaaaa 960

tatttgttgt cggaggatac cagcgggaaa ttcagcgtcg ataagctgaa attcgacaaa 1020

ttgtataaaa tgctgaccga gatttacacc gaggataact tcgtcaagtt ttttaaggtg 1080

ttaaatgcga agaccttttt aaactttgat aaagcggtgt ttaaaattaa tatcgtgccg 1140

aaggtgaatt acaccatcta cgatggtttc aatttacgca acacgaatct ggcggcgaat 1200

tttaatggcc aaaacaccga aattaacaac atgaacttta cgaagttaaa gaatttcacg 1260

ggcttattcg aattctacaa gttattatgc gtgcgcggca tcattaccag caaggcaggt 1320

gcgggcaagt ccttggttcc gcgtggcagc gccggcgccg gcgcgctcaa tgatctgtgt 1380

attaaagtca ataactggga cctgttcttc agcccgagcg aggataactt taccaacgac 1440

ttaaacaaag gcgaggagat cacgagcgat acgaacatcg aggcggcgga ggaaaatatt 1500

agcctggacc tcattcagca gtactatctg acgttcaatt ttgacaatga gccggagaac 1560

atcagcattg aaaatctcag cagcgacatc atcggtcagt tggaactgat gccgaacatt 1620

gaacgctttc cgaacggcaa aaaatatgaa ctggacaagt ataccatgtt ccattactta 1680

cgcgcacagg aatttgagca cggcaagagc cgcattgcgc tgaccaatag cgttaacgag 1740

gccttgttaa atccgagccg tgtctacacg ttcttcagca gcgattatgt caaaaaagtg 1800

aacaaggcga ccgaagccgc gatgtttttg ggctgggtcg agcaattggt ttacgatttt 1860

accgacgaaa ccagcgaggt gagcacgacc gacaaaattg cagatatcac catcatcatt 1920

ccgtacatcg gtccggcgct caatatcggc aatatgttat acaaggacga ctttgtgggc 1980

gcgctgatct ttagcggcgc ggttatctta ttagaattca tcccggagat cgcaatcccg 2040

gtcttgggca cctttgcgtt ggtgagctat atcgcgaata aagtgctcac ggtccaaacc 2100

atcgataacg cgctcagcaa gcgtaatgag aaatgggacg aggtttataa gtatatcgtg 2160

accaactggt tagcaaaagt caatacgcag atcgatctca tccgcaaaaa aatgaaagaa 2220

gccttggaaa atcaagcgga ggcaaccaaa gccatcatta attaccagta taaccaatat 2280

accgaagaag aaaaaaacaa tatcaacttc aatatcgatg atttgagcag caaactgaac 2340

gagagcatta acaaagcgat gattaacatc aacaagttct tgaatcaatg cagcgtgagc 2400

tatctcatga acagcatgat cccgtatggc gtcaaacgct tggaagattt tgacgccagc 2460

ctgaaagatg cgctcctcaa gtatatttat gacaaccgcg gcaccctcat tggccaggtg 2520

gaccgcttga aggataaagt gaacaatacg ctcagcacgg atatcccgtt ccagctgagc 2580

aagtacgtcg acaaccagcg cttactgagc acctttaccg agtatatcaa gaacatcatt 2640

aataccagca tcctcaactt gcgctatgag agcaatcacc tgatcgacct cagccgctac 2700

gccagcaaga tcaacatcgg cagcaaggtc aatttcgacc cgatcgataa gaatcagatc 2760

caattgttta acctggaaag cagcaagatc gaggttatct tgaagaacgc gattgtgtac 2820

aacagcatgt atgagaactt tagcaccagc ttctggattc gtatcccgaa atatttcaac 2880

agcattagcc tgaacaacga gtacaccatt atcaactgca tggaaaacaa cagcggttgg 2940

aaggtgagcc tgaactacgg cgagattatc tggaccctgc aggacaccca agaaatcaag 3000

cagcgtgtgg ttttcaagta cagccaaatg atcaacatca gcgattacat taaccgttgg 3060

atctttgtta ccattaccaa caaccgtctg aacaacagca aaatttacat caacggtcgt 3120

ctgatcgacc agaagccgat tagcaacctg ggcaacatcc acgcgagcaa caacattatg 3180

ttcaagctgg acggttgccg tgatacccac cgttatattt ggatcaagta cttcaacctg 3240

ttcgataagg agctgaacga gaaggaaatc aaagacctgt atgataacca gagcaacagc 3300

ggtattctga aagacttctg gggcgattac ctgcaatatg acaagccgta ttacatgttt 3360

aacgcgggta acaagaacag ctacatcaaa ctgaagaaag atagcccggt gggtgaaatt 3420

ctgggtccgc gtggcagcgt tatgaccacc aacatctatc tgaacagcag cctgtaccgt 3480

ggcgaaaagt tcattatccg tcgtaaaagc aacagccaga gcatcaacga cgatattgtg 3540

cgtaacgagg actatatcta cctggatttc tttaacctga accaagaatg gcgtgtttac 3600

acctacaagt acttcaagaa agaagaggaa aagctgtttc tggcgccgat tagcgacagc 3660

gatgaattct ataacaccat tcagatcaaa gagtacgacg aacagggtac caacagctgc 3720

caactgctgt ttaagaaaga cgaggaaagc accgatgaga tcggtctgat tggcatccac 3780

cgtttttacg aaagcggcat cgtgttcgag gaatacaagg attacttctg catcagcaag 3840

tggtatctga aagaggttaa gcgtaaaccg tacaacctga aactgggctg caactggcaa 3900

tttattccgg tggatgatgg ctggggtgaa cgtccactag tgccacgcgg ttccgcgaat 3960

tcgagctccg tcgacaagct ttggagccac ccgcagttcg aaaaactcga gcaccaccac 4020

caccaccact ga 4032

<210>14

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> hSytI peptide

<400>14

Gly Glu Gly Lys Glu Asp Ala Phe Ser Lys Leu Lys Glu Lys Phe Met

1 5 10 15

Asn Glu Leu His Lys

20