WO2022179552A1 - 一种经修饰的神经毒素的单链多肽及其用途 - Google Patents

一种经修饰的神经毒素的单链多肽及其用途 Download PDF

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WO2022179552A1
WO2022179552A1 PCT/CN2022/077631 CN2022077631W WO2022179552A1 WO 2022179552 A1 WO2022179552 A1 WO 2022179552A1 CN 2022077631 W CN2022077631 W CN 2022077631W WO 2022179552 A1 WO2022179552 A1 WO 2022179552A1
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chain polypeptide
polypeptide
protease
amino acid
chain
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French (fr)
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张岩
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重庆誉颜制药有限公司
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Priority to KR1020237031001A priority Critical patent/KR20230144618A/ko
Priority to EP22758916.5A priority patent/EP4289868A1/en
Priority to US18/547,979 priority patent/US20240226250A9/en
Publication of WO2022179552A1 publication Critical patent/WO2022179552A1/zh

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    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
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    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • A—HUMAN NECESSITIES
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    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24068—Tentoxilysin (3.4.24.68), i.e. tetanus neurotoxin
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    • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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    • A61K2800/86—Products or compounds obtained by genetic engineering
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    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a single-chain polypeptide of a modified neurotoxin and use thereof.
  • Neurotoxins including, for example, botulinum toxin (BoNTs) and tetanus toxin (Tenanustoxin, Clostridiumtenani, tetX or TeNT), belong to Clostridium neurotoxin (CNTs, hereinafter referred to as Clostridium neurotoxin), It is a typical highly potent neurotoxin with lethal doses in humans ranging from 0.1 ng to 1 ng per kilogram of body weight (Tonello et al., Adv. Exp. Med. & Biol. 389:251-260 (1996)). Clostridial neurotoxins bind specifically to nerve cells and disrupt neurotransmitter release.
  • BoNTs botulinum toxin
  • TeNT tetanus toxin
  • Clostridium neurotoxin Clostridium neurotoxin
  • botulinum neurotoxin BoNTs, botulinum toxin
  • tetanus toxin tetX
  • BoNTs botulinum neurotoxin
  • tetX tetanus toxin
  • L A light chain
  • H heavy chain
  • the light chain contains a protease activity domain
  • the heavy chain contains a translocation domain (N-terminal) and a receptor binding domain (C-terminal).
  • the holotoxin is synthesized in the body as a single chain, and the single-chain polypeptide containing the heavy chain is subsequently cleaved in a post-translational modification, a process that involves proteolysis of an exposed region called the loop region, resulting in the final formation of the L-chain and H-chain, Two active neurotoxins linked by disulfide bonds.
  • Botulinum neurotoxins (BoNTs) of all serotypes and the related tetanus neurotoxin (TexT) secreted by Clostridium tetani are Zn 2+ proteases that prevent synapses by cleaving proteins involved in the formation of the SNARE complex that controls cell membrane fusion Exocytosis, inhibits neurotransmitter release, and cuts off nerve signal transmission.
  • botulinum neurotoxin inhibits the release of excitatory transmitters in peripheral neurons, causing muscle paralysis, while tetanus neurotoxin inhibits the release of inhibitory transmitters in central neurons, causing muscle spasm.
  • CNTs activity has been shown to affect glandular secretion.
  • Botulinum neurotoxin type A (BoNT/A) was approved in the United States in 1989 for the treatment of strabismus, blepharospasm and other disorders.
  • botulinum toxin is injected directly into the muscle to be treated in a complex with additional bacterial proteins, and at physiological pH, the toxin is released from the protein complex (Eisele et al. 2011, Toxicon 57 (Eisele et al. 2011, Toxicon 57( 4): 555-65), and produced the expected pharmacological effects.
  • the modified BoNT/A formulations do not contain complexed proteins. Because the effects of BoNT are only temporary, patients need to take BoNT repeatedly to maintain the therapeutic effect.
  • the FDA also approved botulinum toxin type B for the treatment of cervical dystonia.
  • US Patent No. 6,113,915 and US Patent No. 5,721,215 disclose applications for the treatment of pain;
  • US Patent No. 5,053,005 discloses the use of botulinum neurotoxin in the treatment of Application of neuromuscular diseases;
  • Elston, J.S., etc. disclose the application of BoNT/A in the treatment of strabismus (British Journal of Ophthalmology, 1985, 69, 718-724 and 891-896);
  • Adenis, J.P. Application in the treatment of blepharospasm Ophthalmol., 1990, 13(5), 259-264
  • BoNT/A disclosed the application of BoNT/A in the treatment of spastic oromandibular dystonia torticollis (Neurology, 1987, 37, 616-623); Blitzer et al. disclosed the use of BoNT/A to treat spastic dysphonia (Ann. Otol. Rhino. Laryngol, 1985, 94, 591-594); Brin et al. disclosed that BoNT/A was used in the treatment of tongue tension. Use in disorders (Adv. Neurol. (1987) 50, 599-608) Treatment with BoNT/A; Cohen et al. disclose the use of BoNT/A in the treatment of writing spasm (Neurology (1987) 37 (Suppl 1), 123 -4).
  • botulinum neurotoxin has definite medical and cosmetic efficacy
  • the industrial production of this toxin faces many difficulties, and the problems faced mainly focus on the two aspects of purity and safety (low toxicity or no toxicity).
  • Clostridial neurotoxins are linked to each other by disulfide bonds. Between the participating cysteine residues there is a linker region or loop region (loop, also defined in the industry as a linker sequence or loop sequence), the length of which varies widely among the various serotypes of Clostridial neurotoxins big.
  • This loop is cleaved by a as yet uncharacterized Clostridial endopeptidase at the latest when toxin is released from Clostridium during cell lysis, with the ratio of cleaved and uncleaved species varying between serotypes.
  • neurotoxins can be expressed as inactive (or low-active) single-chain forms, preserving the solubility of protein chains, reducing protein misfolding and its resulting susceptibility to protease attack, and improving toxin production, is a comparative actual practice.
  • One approach is to modify sites in the neurotoxin's loop structure to be recognized and hydrolyzed only by specific proteases.
  • Patent US7709228 discloses enzyme cleavage sites that can be modified and proteases that can specifically recognize these sites.
  • the EK enzyme cleavage site is used to modify the ring region of TeTx in a specific embodiment, but the toxicity of its recombinant product is equivalent to two-fifths of that of natural TeTx, and its decline is not very significant.
  • the patent also compares the proteolytic activity and toxicity of uncleaved recombinant BoNT/E and native BoNT/E. Although uncleaved recombinant BoNT/E has significantly lower activity, its toxicity is not greatly reduced.
  • the present invention provides a modified neurotoxic single-chain polypeptide and its application, specifically as follows:
  • a first aspect of the present invention provides a single-chain polypeptide of a modified neurotoxin, the single-chain polypeptide comprising:
  • a first polypeptide fragment comprising:
  • a second functional amino acid structural region comprising a receptor binding domain that can bind to target cell surface receptors and/or a translocation domain that can mediate polypeptide translocation across vesicle membranes.
  • tagged protein refers to a class of protein molecules that can bind to a specific ligand.
  • the tag protein is selected from tag proteins known to those skilled in the art or tag proteins designed by computer programs, and the tag proteins can specifically bind to known substrates.
  • the tag protein is selected from but not limited to the following proteins: glutathione S-transferases (GSTs), C-myc, chitin binding domain, maltose binding protein (MBP), SUMO heterologous Affinity moiety, monoclonal antibody or protein A, streptavidin binding protein (SBP), cellulose binding domain, calmodulin binding peptide, S tag, Strep tag II, FLA, protein A, protein G, group Amino acid affinity tag (HAT), polyhistidine.
  • GSTs glutathione S-transferases
  • C-myc C-myc
  • chitin binding domain chitin binding domain
  • MBP maltose binding protein
  • SBP streptavidin binding protein
  • calmodulin binding peptide S tag
  • Strep tag II FLA
  • protein A protein G
  • protein G group Amino acid affinity tag
  • polyhistidine polyhistidine
  • the "tag protein” can increase the solubility of the toxin polypeptide precursor in the host.
  • the "tag protein” allows the toxin polypeptide precursor to exist in the host cell in a soluble manner.
  • the tag protein is located at the N-terminus of the single-chain polypeptide.
  • the tag protein is glutathione s-transferase (GSTs), more preferably, the amino acid sequence of the glutathione s-transferase is shown in SEQ ID NO: 2 .
  • neither the first protease cleavage site nor the second protease cleavage site can be cleaved by human protease or the protease produced by the host cell expressing the single-chain polypeptide itself.
  • first protease cleavage site and the second protease cleavage site are the same or different.
  • the first protease cleavage site and the second protease cleavage site are not recognized and cleaved by endogenous proteases in the host cell when expressed or when used.
  • the specific protease is selected from but not limited to one or a combination of the following proteases: non-human enterokinase, tobacco etch virus protease, protease derived from Bacillus subtilis, derived from Bacillus amyloliquefaciens protease from rhinovirus, papain, homologue of insect papain or homologue of crustacean papain.
  • the protease that specifically recognizes the first protease and the protease that specifically recognizes the cleavage site of the second protease are both proteases derived from rhinovirus.
  • the second enzyme cleavage site inserts, partially replaces or completely replaces the naturally occurring loop region between the first functional peptide segment and the second functional peptide segment.
  • the insertion refers to inserting between certain two amino acids in the loop region;
  • the partial replacement refers to that the second enzyme cleavage site replaces part of the amino acid sequence of the loop region ;
  • Said complete replacement means that the amino acid sequence of the natural loop region is completely replaced by the second enzyme cleavage site.
  • the first protease cleavage site and the second protease cleavage site are selected from but not limited to one or a combination of the following enzyme cleavage sites: DDDDK, EXXYXQS/G, HY, YH or LEVLFQGP .
  • the first protease cleavage site and the second protease cleavage site are both LEVLFQGP.
  • the connecting short peptide does not exceed 5 amino acids.
  • the linker peptide is capable of making the first protease cleavage site more readily recognized or bound by its protease.
  • the linking peptide does not affect the function of the single-chain polypeptide.
  • the connecting short peptide remaining at the N-terminus of the second polypeptide fragment will not affect the function of the second polypeptide fragment.
  • the GS part of the connecting short peptide does not exceed 5 amino acid residues, more preferably, the connecting short peptide is selected from glycine-serine (Glycine-Serine, referred to as GS) short peptide, GGS, GGGS, GGGGS, GSGS, GGSGS, GSGGS, GGSGS, GGGSS, etc. linking short peptides.
  • Glycine-Serine Glycine-Serine
  • amino acid sequence of the structural region comprising the first protease cleavage site and the connecting short peptide is LEVLFQGPLGS.
  • the metal ion-dependent protease activity domain is a Zn 2+ -dependent protease activity domain.
  • the first functional amino acid structural region and/or the second functional amino acid structural region are encoded by natural sequences and/or artificially synthesized sequences. More preferably, the first functional amino acid structural region of the single-chain polypeptide comprises the Zn 2+ protease activity domain of the light chain of Clostridial toxin.
  • the target cell capable of binding the receptor binding domain of the human cell surface antigen in the second functional amino acid structural region refers to the target cell in which the SNARE complex exists.
  • the target cell in which the SNARE complex exists For example: nerve cells, pancreatic cells or other cells that have SNARE complexes.
  • the target cells in the second functional amino acid structural region are human nerve cells or pancreatic cells
  • the receptor binding domain is a receptor binding domain capable of specifically binding human nerve cells or pancreatic cells.
  • the receptor binding domain of the second functional amino acid structural region is the cell surface antigen binding domain of the heavy chain of Clostridium toxin, and the mediating polypeptide of the second functional amino acid structural region crosses the vesicle membrane.
  • the transferred translocation domain is the domain that mediates the transfer of Clostridial toxins across the vesicle membrane.
  • Clostridial toxin is botulinum toxin or tetanus toxin.
  • the botulinum toxin is selected from any of the serotypes BoNT/A-BoNT/H and their derivatives known in the prior art.
  • the Clostridial toxin is selected from any one of tetanus toxin or its derivatives. More preferably, the first functional amino acid structural region comprises BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H or tetanus toxin. Part or all of the light chain.
  • the second functional amino acid structural region comprises BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H or tetanus toxin. part or all of the heavy chain.
  • the first functional amino acid structural region is the light chain of BoNT/A
  • the second functional amino acid structural region is the heavy chain of BoNT/A
  • the first functional amino acid structural region and the second functional amino acid structural region can be from different serotypes.
  • amino acid sequence of the second polypeptide fragment comprises the fragment shown in SEQ ID NO:7.
  • the single-chain polypeptide comprises, starting from the N-terminus, glutathione S-transferase, LEVLFQGPLGS, the light chain of BoNT/A, the LEVLFQGP and the heavy chain of BoNT/A.
  • amino acid sequence of the single-chain polypeptide is shown in SEQ ID NO: 11.
  • the second aspect of the present invention provides a nucleic acid molecule encoding the above single-chain polypeptide.
  • the nucleic acid molecule consists of nucleotide sequences encoding various parts of the single-chain polypeptide.
  • sequence encoding the tag protein comprises the sequence shown in SEQ ID NO: 1.
  • the nucleotide sequence encoding the structural region comprising the first protease cleavage site comprises the sequence shown in SEQ ID NO:3.
  • nucleotide sequence encoding the structural region comprising the second protease cleavage site comprises the sequence shown in SEQ ID NO:8.
  • amino acid sequence encoding the second polypeptide fragment comprises the sequence shown in SEQ ID NO:7.
  • the nucleotide sequence encoding the second polypeptide fragment comprises the sequence shown in SEQ ID NO:6.
  • nucleotide sequence encoding the single-chain polypeptide is shown in SEQ ID NO: 10.
  • the third aspect of the invention provides a vector, the vector comprising the above-mentioned nucleic acid molecule or an open reading frame encoding the above-mentioned single-chain polypeptide.
  • the vectors are plasmids, bacteriophages, viral vectors and the like.
  • the fourth aspect of the invention provides a cell comprising the above-mentioned vector or expressing the above-mentioned single-chain polypeptide.
  • the cells are eukaryotic cells or prokaryotic cells.
  • the cells are selected from Escherichia coli, yeast, cyanobacterial or mammalian cells, insect cells, plant cells or amphibian cells.
  • the cells are Escherichia coli.
  • the fifth aspect of the present invention provides a method for preparing the above single-chain polypeptide, the method comprising using any one of the above nucleic acids, vectors or cells to prepare the single-chain polypeptide.
  • the method comprises: (1) designing a nucleic acid molecule encoding the single-chain polypeptide; (2) constructing a vector comprising the nucleic acid molecule; (3) transforming the nucleic acid vector into a suitable host cell; (4) culturing the host cell, and allowing or inducing the host cell to express the single-chain polypeptide encoded by the nucleic acid vector.
  • the method further comprises (5) lysing the cells expressing the single-chain polypeptide to obtain a cell lysate comprising the single-chain polypeptide.
  • the sixth aspect of the present invention provides a single-chain polypeptide derivative, the single-chain polypeptide derivative comprising:
  • a second functional amino acid structural region comprising a receptor binding domain that can bind to target cell surface receptors and/or a translocation domain that can mediate polypeptide translocation across vesicle membranes.
  • the metal ion-dependent protease activity domain is a Zn 2+ -dependent protease activity domain.
  • the target cells are human nerve cells, pancreatic cells or other cells with SNARE complexes.
  • the single-chain polypeptide derivative is a product obtained after the first protease cleavage site of the single-chain polypeptide is cleaved.
  • the length of the connecting short peptide is no more than 5 amino acids.
  • a seventh aspect of the present invention provides a method for preparing the above-mentioned single-chain polypeptide derivative, the method comprising obtaining any one of the above-mentioned single-chain polypeptide by cleaving the first protease cleavage site by a first protease.
  • the eighth aspect of the present invention provides a method for preparing a toxin polypeptide, the preparation method comprising: cleaving the above-mentioned single-chain polypeptide by a first protease to remove a tagged protein, and cleaving a first functional amino acid by a second protease
  • the structural region and the second functional amino acid structural region form at least one dimer.
  • the first functional amino acid structural region and the second functional amino acid structural region are connected to form a dimer structure through a disulfide bond.
  • the ninth aspect of the present invention provides a toxin polypeptide, the toxin polypeptide comprises:
  • a second amino acid structural region comprising a receptor binding domain that can bind to a receptor on the target cell surface and/or a translocation domain that can mediate the translocation of the polypeptide across the vesicle membrane.
  • Said (a), (b) are not connected by a peptide bond, preferably by a disulfide bond.
  • a dimer structure is formed between (a) and (b) of the toxin polypeptide.
  • amino acid residues cleaved by the second protease are included between (a) and (b) of the toxin polypeptide.
  • amino acid sequence of the toxin polypeptide comprises the sequence shown in SEQ ID NO:7.
  • the toxin polypeptide is obtained by the preparation method of the above toxin polypeptide.
  • the above-mentioned toxin polypeptide precursors (including single-chain polypeptides or single-chain polypeptide derivatives) have low toxicity relative to the highly toxic toxin polypeptides after activation, and more preferably, the low toxicity is relative to the high toxicity after activation
  • the activity of the activated toxin polypeptide is at least 5000 times higher than that of the toxin polypeptide precursor, and further, the activity of the activated toxin polypeptide is higher than that of the toxin polypeptide precursor by 6000, 7000, 8000, 9000, 10000, 12000, 15000 times, or even higher.
  • the tenth aspect of the present invention provides a composition comprising the above-mentioned modified single-chain polypeptide, single-chain polypeptide derivative or toxin polypeptide.
  • the composition further comprises excipients or diluents. Further preferably, the composition is substantially free of trypsin.
  • the composition is a pharmaceutical composition.
  • the eleventh aspect of the present invention provides the use of the above single-chain polypeptide, single-chain polypeptide derivative or toxin polypeptide in the preparation of a composition.
  • the composition is a pharmaceutical composition that treats a related disease by interfering with neurotransmitter release
  • the disease includes neuromuscular disease, autonomic disorder or pain, a symptom of the disease Including: spastic dysphonia, spastic torticollis, laryngeal dystonia, oromandibular dysphonia, tongue dystonia, cervical dystonia, focal dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, Cerebral palsy, focal spasticity and other speech disorders, spastic colitis, neurogenic bladder, pelvic floor achalasia, limb spasms, convulsions, tremors, bruxism, anal fissures, achalasia, dysphagia, and other muscle tone Disorders and other disorders characterized by involuntary movement of muscle groups, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, secretion disturbances,
  • the twelfth aspect of the present invention provides non-therapeutic uses of the above-mentioned single-chain polypeptide, single-chain polypeptide derivatives, and toxin polypeptides in improving related symptoms by interfering with neurotransmitter release.
  • the use is cosmetic, such as reducing facial wrinkles, shrinking facial muscles to achieve a slimming effect, and the like.
  • the single-chain polypeptide provided by the present invention makes the tag protein easier to be cleaved by adding a short linking peptide, so that the single-chain polypeptide has a higher yield and a higher purity.
  • the single-chain polypeptide involved in the present invention its tag protein makes the protease domain of the first functional amino acid structural region unable to function as a protease.
  • the site is replaced with a site that can only be recognized by a specific enzyme, so that the molecular activity (neurotoxicity) of the single-chain polypeptide is reduced to less than 1/10,000 of that of a natural toxin, thus dividing the steps of producing the activation molecule into the described
  • the production of the single-chain polypeptide and the activation of the single-chain polypeptide, the activation of the single-chain polypeptide refers to contacting it with a specific protease, cleaving the peptide bond between the two functional peptide segments, making it active molecule.
  • the production of single-chain polypeptide occupies most of the production process, however, because its toxicity is greatly reduced, the risk in the production process is reduced, and the entire industrial production is easier to carry out.
  • the tag protein in the present invention refers to a heterologous affinity molecule, which can bind to a specific ligand, and then detach from the ligand, so that the molecule co-expressed with the tag protein is easier to obtain from different polypeptides. or protein products are selected.
  • the single-chain polypeptide produced by the present invention has an altered target-specific binding region or an altered active region, or both, compared with naturally derived neurotoxins.
  • the modified neurotoxin-activatable single-chain polypeptide provided by the present invention is easier to produce and purify than the prior art, and can be used for clinical use. Large-scale production of properly folded and highly purified toxin polypeptides.
  • Figure 1 SDS-PAGE results of preliminary purification of GSTs-BoNT/A and further removal of GSTs tags, wherein the first lane is GSTs-BoNT/A obtained after preliminary purification by GSTs affinity chromatography column, and the second lane is preliminary purified GSTs-BoNT/A
  • the protein was digested with Rinovirus 3C Protease to remove the GSTs-tagged protein to obtain BoNT/A after GSTs were removed.
  • Figure 2 SDS-PAGE results of highly purified BoNT/A protein obtained by further purification of GSTs-tagged product through ion exchange column.
  • Figure 3 Validation results of double-strand dissociation of BoNT/A under reducing conditions.
  • Example 1 Design and construction of nucleic acid molecules encoding single-chain polypeptides of modified neurotoxins:
  • Nucleic acid molecules include sequentially from the 5' end:
  • nucleotide sequence encoding a second polypeptide fragment comprising the nucleotide sequence of the BoNT/A light chain, the second protease cleavage site and the BoNT/A heavy chain, as shown in SEQ ID NO: 6,
  • the encoded amino acid sequence is shown in SEQ ID NO: 7; wherein, the nucleotide sequence encoding the second protease cleavage site is shown in SEQ ID NO: 8, and the encoded amino acid sequence is shown in SEQ ID NO: 9 ;
  • the nucleotide sequence encoding the single-chain polypeptide is shown in SEQ ID NO: 10, and the amino acid sequence of the encoded single-chain polypeptide is shown in SEQ ID NO: 11.
  • Example 2 Construction of a plasmid comprising the nucleic acid molecule of Example 1
  • GSTs-BoNT/A optimized by genetic engineering was artificially synthesized in Example 1, and NdeI and NotI restriction sites were added synthetically at both ends, and NdeI and NotI were digested at 37 degrees Celsius (New England Biolabs), using QIquick gel extraction kit (Qiagen). ) was purified and inserted into the pET28a (Novagen) plasmid vector at the NdeI and NotI sites using T4 DNA ligase (NEB).
  • Example 3 Transfection of the plasmid constructed in Example 2 into host cells
  • composition and ratio of medium 11.8g/L tryptone, 23.6g/L yeast extract, 9.4g/L K 2 HPO 4 2.2g/L KH 2 PO 4 4ml/L glycerol.
  • Culture conditions 37°C overnight shaking at 250rpm to culture cells.
  • the OD 600 can be selected from 0.2-1.5, the temperature can be selected from 37°C to 10°C, and the expression time can be 5-16 hours.
  • Harvest cells harvest E. coli by centrifugation at 3000 rpm for 30 minutes.
  • GSTs-BoNT/A use GenScript glutathione purification resin to prepare a glutathione purification resin chromatography column with a diameter of 1 cm and a height of 1 cm, and slowly pour 25 ml of lysate onto the upper layer of the column. Be careful not to wash up the resin, and slowly pass through the column to adsorb the GSTs-BoNT/A in the lysate after one hour. GSTs-BoNT/A was obtained by elution according to conventional methods.
  • the general procedure is as follows: the column is washed with 20 column volumes of phosphate buffer, and 10 column volumes of freshly prepared 10 mM glutathione elution buffer (0.154 g of reduced glutathione dissolved in 50 ml of 50 mM Tris- GSTs-BoNT/A was eluted with HCl (pH 8.0), and the elution of the fusion protein was monitored using the absorbance reading at 280 nm. The eluted protein was excised under the action of Rinovirus 3C Proteas to the GSTs-tagged protein.
  • the purified GSTs-BoNT/A was separated by electrophoresis at 200 volts using 4-12% SDS-PAGE (Biorad). The main band with a molecular weight of 175 kd was GSTs-BoNT/A.
  • Example 5 The product of Example 5 was subjected to the removal of GSTs-tagged proteins and purification of toxic polypeptides:
  • the GSTs-BoNT/A re-adsorbed on the glutathione purification resin chromatography column was treated with GenScript's 3C enzyme. Under the action of the 3C enzyme, the first cleavage site between GSTs and BoNT/A was cleaved On, the GSTs are detached, and the second enzyme cleavage site between the light and heavy chains of BoNT/A is cleaved.
  • the GSTs-removed product was subjected to a conventional SDS-PAGE experiment. As shown in Figure 2, there was no GSTs-tagged protein in the obtained band, indicating that the GSTs-tagged protein had been completely removed. The pictures were scanned by SDS-PAGE, and the obtained BoNT/A was more than 90% pure as calculated from the gray density of the bands.
  • the effect is similar to that of GS, and the tagged protein can be well exposed, so that it can be completely excised.
  • Example 7 Dissociation of duplexes of dimeric BoNT/A under reducing conditions
  • Example 6 The product of Example 6 was subjected to reduction experiments:
  • Example 6 The samples were reduced by treatment with 100 mM dithiothreitol at 100 degrees Celsius for five minutes, and the samples were separated by 4-12% SDS-PAGE (Biorad) electrophoresis at 200 volts to separate heavy and light chains.
  • SDS-PAGE Biorad electrophoresis at 200 volts to separate heavy and light chains.
  • the product obtained in Example 6 was reduced under reducing conditions, and the obtained product was subjected to a conventional SDS-PAGE experiment to obtain two different bands with molecular weights of 100Kda and 50Kda respectively, proving that Example 6
  • the resulting product is a dimer structure in which two peptide fragments are linked by disulfide bonds.
  • the LD 50 of intraperitoneal injection of GSTs-BoNT/A obtained in Example 5 into mice is between 45ng-450ng; considering the purity of the injected botulinum toxin protein, the converted LD 50 is between 22.5ng-225ng, the median value is 123.75ng.
  • the LD 50 of BoNT/A obtained in Example 5 by intraperitoneal injection in mice was between 0.02ng and 0.05ng. Considering the purity of the injected botulinum toxin protein, the converted LD50 was between 0.006ng and 0.015ng, with a median value of 0.0105ng (see Table 1).
  • GSTs-BoNT/A has the activity of botulinum toxin.
  • its median lethal dose (LD 50 ) is about 11786 times higher than the protein LD 50 of BoNT/A, indicating that GSTs-BoNT/A recombinant protein toxin polypeptide
  • the bulk molecular activity is about 11786 times weaker than the final product BoNT/A.
  • This experiment proves that the GSTs-BoNT/A recombinant protein toxin polypeptide precursor molecule has the activity of botulinum toxin. Due to the high toxicity of botulinum toxin, a high degree of safe handling is required before the precursor molecule is activated during the production process.
  • the present invention exemplifies the construction of nucleic acid molecules comprising GSTs, a first protease cleavage site, a linking short peptide, and a light chain of BoNT/A, a second protease cleavage site and a heavy chain of BoNT/A; further construction of the nucleic acid comprising the nucleic acid Molecular plasmid capable of expressing GSTs-BoNT/A in E.coli; E.coli transfected with this plasmid expresses single-chain GSTs-BoNT/A, and the single-chain polypeptide has been proved to be the final result of its activation.
  • the toxicity of the product is less than 1/10,000 of that of BoNT/A; after protein purification and elution of GSTs, it is proved that due to the addition of the linking polypeptide, the enzyme cleavage site is easier to be recognized and cleaved by the protease than the short peptide without linking; due to the first protease
  • the cleavage site is the same as the second protease cleavage site, so when GSTs are removed, the second protease cleavage site is also cleaved, and the peptide bond between the first functional amino acid structural region and the second functional amino acid structural region is broken , linked by a disulfide bond to form active BoNT/A, which was shown to have toxicity equivalent to that of natural products.

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Abstract

本发明涉及一种经修饰的神经毒素的单链多肽及其用途，所述单链多肽包含标签蛋白，连接短肽等。所述连接短肽有利于标签蛋白的切割，从而提高单链多肽的纯化，同时，该单链多肽经过活化具有神经毒性，但其本身只有轻微的毒性，在大幅度提高单链多肽得率的同时也提高了生产的安全性和降低了生产工艺的成本。

Description

一种经修饰的神经毒素的单链多肽及其用途
优先权
本申请要求申请号为202110217647.X、申请日为2021年2月26日作为本申请的优先权，优先权文本的内容在此整体引用作为参考。
技术领域
本发明属于生物技术领域，具体涉及一种经修饰的神经毒素的单链多肽及其用途。
背景技术
神经毒素，包括例如肉毒杆菌神经毒素(Botuliumtoxin，简称BoNTs)和破伤风毒素(Tenanustoxin，Clostridiumtenani，简称tetX或TeNT)，都属于梭状芽胞杆菌神经毒素(CNTs，以下简称梭菌神经毒素)，是典型的高效神经毒素，对人的致死剂量在每千克体重0.1ng到1ng之间(Tonello et al.，Adv.Exp.Med.&Biol.389：251-260(1996))。梭菌神经毒素能够特异性地结合神经细胞并破坏神经递质的释放。
肉毒梭菌分泌7种抗原不同的血清型，这些血清型被设计为A到G型肉毒杆菌神经毒素(BoNTs)。肉毒杆菌神经毒素(BoNTs，肉毒素)和破伤风毒素(tetX)虽然同源性仅有65％，但分子结构和分子质量几乎相同，均包含两个由二硫键连接的氨基酸链：一条约50KDa的轻链(L)和一条约100KDa的重链(H)，轻链包含蛋白酶活性域，重链包含易位域(N端)和受体结合域(C端)。全毒素在体内以单链合成，包含重链的单链多肽随后在翻译后修饰中被切割，其过程涉及一个称为环状区的暴露区域被蛋白质水解，最终形成包含L链和H链，二者以二硫键连接的活性神经毒素。所有血清型的肉毒杆菌神经毒素(BoNTs)以及破伤风梭菌分泌的相关破伤风神经毒素(TexT)都是Zn 2+蛋白酶，通过切割参与形成控制细胞膜融合的SNARE复合体的蛋白质阻止突触胞吐，抑制神经递质释放，切断神经信号传递。其中，肉毒杆菌神经毒素抑制外周神经元兴奋性递质释放，致肌肉麻痹，而破伤风神经毒素抑制中枢神经元抑制性递质释放，致肌肉痉挛。此外，CNTs活性已被证明能影响腺体分泌。
A型肉毒杆菌神经毒素(BoNT/A)1989年在美国被批准用于治疗斜视、眼睑痉挛和其他疾病。在一些应用中，肉毒素与额外的细菌蛋白质以复合物形式直接 被注射到待治疗的肌肉中，在生理pH值下，毒素从蛋白质复合物中释放出来(Eisele et al.2011，Toxicon 57(4)：555-65)，并产生预期的药理作用。另一些应用中，被改进BoNT/A制剂不含复合蛋白。由于BoNT的作用只是暂时的，因此患者需要反复服用BoNT以维持治疗效果。另外FDA还批准了B型肉毒杆菌毒素用于治疗颈部张力障碍。
在用肉毒杆菌神经毒素进行各种治疗的应用中，美国专利6,113,915和美国专利5，721，215公开了有关用于治疗疼痛的应用；美国专利第5,053,005号公开了肉毒杆菌神经毒素在治疗神经肌肉疾病的应用；Elston，J.S.等公开了BoNT/A在治疗斜视方面的应用(British Journal of Ophthalmology，1985，69，718-724和891-896)；Adenis，J.P.等公开了BoNT/A在治疗睑痉挛方面的应用(Ophthalmol.，1990，13(5)，259-264)；Jankovic等公开了BoNT/A在治疗痉挛性口下颌张力障碍性斜颈方面的应用(Neurology，1987，37，616-623)；Blitzer等公开了用BoNT/A进行治疗痉挛性发声困难的实例(Ann.Otol.Rhino.Laryngol，1985，94，591-594)；Brin等公开了BoNT/A在治疗舌张力障碍中的应用(Adv.Neurol.(1987)50，599-608用BoNT/A进行治疗；Cohen等公开了BoNT/A在治疗书写痉挛中的应用(Neurology(1987)37(增刊1)，123-4)。
尽管肉毒杆菌神经毒素有明确的医疗和美容疗效，此毒素的工业生产却面临很多困难，面临的问题主要集中在纯度和安全性(低毒性或无毒性)两个方面。
在纯度方面，为了以足够的数量和可溶的形式获得重组蛋白，尤其是较小的多肽，在许多情况下可以将它们作为融合蛋白或杂合蛋白，例如与谷胱甘肽-S-转移酶或麦芽糖结合蛋白融合或杂合的蛋白在大肠杆菌中表达。此外，市场上有许多借助用于亲和纯化的N-末端或C-末端标签(例如His标签、Strep标签或FLAG标签)来表达所需多肽的表达系统。但是在制药工业中，后续添加蛋白质须证明该蛋白质和其可能存在的污染物在进一步的处理中得以完全去除，而这通常需要相当的花费。因此在提高纯度的同时，如何尽可能地去除标签蛋白是工业化多肽制剂生产的需求。
梭菌神经毒素的重链和轻链彼此通过二硫键相连。在参与的半胱氨酸残基之间有接头区域或环状区域(loop，也被行业内定义为接头序列或环序列)，其长度 在各种血清型的梭菌神经毒素之间变化很大。该环最迟会在细胞溶胞过程中从梭菌释放毒素时，被至今还未定性的梭菌内肽酶切割，其中切割和未切割的种类的比率在血清型之间有所不同。例如，对BoNT/A来所，其环序列VRGI ITSKTKSLDKGYNKALNDL在切割中发生两个酶切位点被蛋白酶切割，并切出一个十肽，被切开的轻链和重链在N末端以及C末端由半胱氨酸残基通过二硫键连接。然而在这个过程中，蛋白酶的切割(在环状区域和重链的末端)是随机的，由此会产生许多不被期望的中间产物，从而增加了纯化的难度，并降低了产品的得率。
在由高毒性引起的安全性方面，有一种在大肠杆菌中分别表达及纯化TeTx和BoNTs的重链和轻链的方法(参见Li，et al.，Biochemistry 33：7014-7020(1994)；Zhou，et al.，Biochemistry，34：15175-15181(1995)。在大肠杆菌中的这些分离的链自身是无毒的，然后再用氧化二硫键结合H和L亚单位形成双链。然而这种方法并不实用，因为首先在没有L链时，分离的H链水溶性很差并且非常容易被蛋白水解降解，其次，体外氧化单独表达和纯化的H和L链产生活性双链的效率不高，并且会产生许多无活性的不正确折叠或氧化的分子产生，从而使得从中分离和纯化出含有正确折叠和氧化的H和L链非常困难。
因此，可以将神经毒素作为无活性(或低活性)单链形式表达，保持蛋白链的溶解性，减少蛋白错误折叠及其导致的对蛋白酶攻击的易感性，以及改进毒素产量，是一种比较实际的做法。一个方法是将神经毒素的环状结构中的位点修饰为只能被特定的蛋白酶识别和水解的位点。专利US7709228公开了可以进行修饰的酶切位点和可以特异地识别这些位点的蛋白酶。该专利在具体实施方式中用EK酶切割位点修饰TeTx的环状区域，但其重组产品的毒性相当于天然TeTx的五之二，其下降幅度并不是十分显著。该专利还对比了未切割的重组BoNT/E与天然BoNT/E的蛋白水解活性及其毒性，虽然未切割的重组BoNT/E具有显著低的活性，但是其毒性并未被大幅降低。
发明内容
为了解决重组神经毒素多肽生产中存在的在提高纯度的同时彻底去除标签蛋白的问题，和降低毒性、安全生产的问题，本发明提供了一种经修饰的神经毒性的单链多肽及用途，具体如下：
本发明的第一方面，提供了一种经修改的神经毒素的单链多肽，所述的单链多肽包含：
(I)第一多肽片段，所述第一多肽片段包含：
(a)标签蛋白，
(b)包含第一蛋白酶切割位点的结构区；
(c)连接短肽，
(II)第二多肽片段，所述第二多肽片段包含：
(d)第一功能氨基酸结构区，其包含金属离子依赖的蛋白酶活性域；
(e)包含第二蛋白酶切割位点的结构区；
(f)第二功能氨基酸结构区，其包含可与靶细胞表面受体结合的受体结合域和/或可以介导多肽跨囊泡膜转移的易位域。
术语“标签蛋白”是指可以与特定的配基结合的一类蛋白分子。
优选的，所述的标签蛋白选自本领域技术人员已知的标签蛋白或者经过计算机程序设计的标签蛋白，所述的标签蛋白能够与已知底物特异结合。
更优选的，所述的标签蛋白选自但不限于以下蛋白：谷胱甘肽S-转移酶(GSTs)、C-myc、几丁质结合结构域、麦芽糖结合蛋白(MBP)、SUMO异源亲和部分、单克隆抗体或蛋白A、链霉亲和素结合蛋白(SBP)、纤维素结合结构域、钙调蛋白结合肽、S标签、Strep标签II、FLA、蛋白A、蛋白G、组氨酸亲和标签(HAT)、多聚组氨酸。
优选的，所述的“标签蛋白”可以增加毒素多肽前体的在宿主中的可溶性。
优选的，所述的“标签蛋白”使得毒素多肽前体以可溶的方式存在于宿主细胞中。
在一个具体实施例中，所述的标签蛋白位于单链多肽的N端。
在一个具体实施例中，所述的标签蛋白为谷胱甘肽s-转移酶(GSTs)，更优选的，所述谷胱甘肽s-转移酶的氨基酸序列如SEQ ID NO：2所示。
优选的，所述的第一蛋白酶切割位点和第二蛋白酶切割位点均不能被人蛋白酶或表达所述单链多肽的宿主细胞自身产生的蛋白酶切割。
更优选的，所述的第一蛋白酶切割位点和第二蛋白酶切割位点相同或不同。
所述的第一蛋白酶切割位点和第二蛋白酶切割位点不被表达时的宿主细胞或使用时机体内源性的蛋白酶识别和切割。
更优选的，所述的特定的蛋白酶选自但不限于以下蛋白酶的一种或两种的组合：非人肠激酶、烟草蚀刻病毒蛋白酶、来源于枯草芽孢杆菌的蛋白酶、来源于解淀粉芽孢杆菌的蛋白酶、来源于鼻病毒的蛋白酶、木瓜蛋白酶、昆虫木瓜蛋白酶的同源物或甲壳动物木瓜蛋白酶的同源物。
在一个具体实施例中，所述的特异地识别第一蛋白酶和所述的特异地识别第二蛋白酶切割位点的蛋白酶均为来源于鼻病毒的蛋白酶。
优选的，第二酶切位点嵌入、部分替换或全部替换第一功能肽段和第二功能肽段之间的天然存在的环状区域。所述的嵌入是指插入在所述的环状区域的某两个氨基酸之间；所述的部分替换是指所述的第二酶切位点替换了所述的环状区域的部分氨基酸序列；所述的完全替换是指天然的环状区域的氨基酸序列完全被第二酶切位点所取代。
更优选的，所述的第一蛋白酶切割位点和第二蛋白酶切割位点选自但不限于以下酶切位点的一种或两种的组合：DDDDK、EXXYXQS/G、HY、YH或LEVLFQGP。
在一个具有实施例中，所述的第一蛋白酶切割位点和第二蛋白酶切割位点均为LEVLFQGP。
优选的，所述的连接短肽不超过5个氨基酸。
优选的，所述连接短肽能够使第一蛋白酶切割位点更容易被其蛋白酶识别或结合。
更优选的，所述的连接短肽不影响单链多肽的功能。
更优选的，所述的第一蛋白酶切割位点被切开后，保留在第二多肽片段N端的连接短肽不会影响第二多肽片段的功能。
更优选的，所述的连接短肽GS部分不超过5个氨基酸残基，更优选的，所述的连接短肽选自甘氨酸-丝氨酸(Glycine-Serine，简称GS)短肽，GGS，GGGS，GGGGS，GSGS，GGSGS，GSGGS，GGSGS，GGGSS等连接短肽。
在一个具体实施例中，所述的包含第一蛋白酶切割位点的结构区和连接短肽的氨基酸序列为LEVLFQGPLGS。
优选的，所述的金属离子依赖的蛋白酶活性域为Zn 2+依赖的蛋白酶活性域。
优选的，所述的第一功能氨基酸结构区和/或第二功能氨基酸结构区由天然序列和/或人工合成的序列编码。更优选的，所述的单链多肽的第一功能氨基酸结构区包含梭菌毒素轻链的Zn 2+蛋白酶活性域。
优选的，所述的第二功能氨基酸结构区中的能够结合人体细胞表面抗原的受体结合域的靶细胞是指存在SNARE复合体的靶细胞。例如：神经细胞、胰腺细胞或其他存在SNARE复合体的细胞。
更优选的，所述的第二功能氨基酸结构区中的靶细胞是指人神经细胞或胰腺细胞，所述的受体结合域为能够特定结合人神经细胞或胰腺细胞的受体结合域。
更优选的，所述的第二功能氨基酸结构区的受体结合域为梭菌毒素的重链的细胞表面抗原结合结构域，所述的第二功能氨基酸结构区的介导多肽跨囊泡膜转移的易位域为介导梭菌毒素跨囊泡膜转移的结构域。
更优选的，所述的梭菌毒素为肉毒杆菌毒素或破伤风毒素。
更优选的，所述的肉毒杆菌毒素选自现有技术已知的血清型BoNT/A-BoNT/H以及它们的衍生物中的任意一种。
更优选的，所述的梭菌毒素选自破伤风毒素或其衍生物中的任意一种。更优选的，所述的第一功能氨基酸结构区包含BoNT/A、BoNT/B、BoNT/C、BoNT/D、BoNT/E、BoNT/F、BoNT/G、BoNT/H或破伤风毒素的轻链的部分或全部。
更优选的，所述的第二功能氨基酸结构区包含BoNT/A、BoNT/B、BoNT/C、BoNT/D、BoNT/E、BoNT/F、BoNT/G、BoNT/H或破伤风毒素的重链的部分或全部。
在一个具体实施例中，所述的第一功能氨基酸结构区为BoNT/A的轻链，所述的第二功能氨基酸结构区为BoNT/A的重链。
更优选的，所述的第一功能氨基酸结构区和第二功能氨基酸结构区可以来自不同的血清型。可以是上述血清型的任意组合，例如，所述的第一功能氨基酸结构区衍生自BoNT/A的轻链，所述的第二功能氨基酸结构区衍生自BoNT/B的重链，或者，所述的第一功能氨基酸结构区衍生自BoNT/A的轻链，所述的第二功能氨基酸结构区衍生自BoNT/C的重链等等。
更优选的，所述的第二多肽片段的氨基酸序列包含SEQ ID NO：7所示的片段。
在一个具体实施例，所述的单链多肽从N端开始依次包含：谷胱甘肽S-转移酶、LEVLFQGPLGS、BoNT/A的轻链、LEVLFQGP和BoNT/A的重链。
更优选的，所述的单链多肽的氨基酸序列如SEQ ID NO：11所示。
本发明的第二方面，提供了一种编码上述单链多肽的核酸分子。
优选的，所述的核酸分子由编码所述的单链多肽的各个部分的核苷酸序列组成。
在一个具体实施例中，所述的编码标签蛋白的序列包含如SEQ ID NO：1所示的序列。
在一个具体实施例中，所述的编码包含第一蛋白酶切割位点的结构区的核苷酸序列包含如SEQ ID NO：3所示的序列。
在一个具体实施例中，所述的编码包含第二蛋白酶切割位点的结构区的核苷酸序列包含如SEQ ID NO：8所示的序列。
优选的，所述的编码第二多肽片段的氨基酸序列包含如SEQ ID NO：7所示的序列。
优选的，所述的编码第二多肽片段的核苷酸序列包含如SEQ ID NO：6所示的序列。
优选的，所述编码单链多肽的核苷酸序列如SEQ ID NO：10所示。
发明的第三方面，提供了一种载体，所述的载体包含上述核酸分子或编码上述的单链多肽的开放阅读框架。
优选的，所述的载体为质粒、噬菌体、病毒载体等等。
发明的第四方面，提供了一种细胞，所述的细胞包含上述的载体或表达上述的单链多肽。
优选的，所述细胞是真核生物细胞或原核生物细胞。
更优选的，所述的细胞选自大肠杆菌、酵母、蓝藻或哺乳动物细胞、昆虫细胞、植物细胞或两栖动物细胞。
更优选的，所述的细胞为大肠杆菌。
本发明的第五方面，提供了一种制备上述单链多肽的方法，所述方法包括利用上述任意一种核酸、载体或者细胞制备所述单链多肽。
进一步，所述的方法包括：(1)设计编码所述单链多肽的核酸分子；(2)构建包含所述核酸分子的载体；(3)转化所述的核酸载体到适宜的宿主细胞中；(4)培养所述宿主细胞，及允许或诱导宿主细胞表达该核酸载体编码的单链多 肽。优选的，所述方法还包括(5)裂解表达所述单链多肽的细胞，获得包含所述单链多肽的细胞裂解产物。
本发明的第六方面，提供了一种单链多肽衍生物，所述单链多肽衍生物包含：
(I)连接短肽
(II)第二多肽片段，所述第二多肽片段包含：
(a)第一功能氨基酸结构区，其包含金属离子依赖的蛋白酶活性域；
(b)包含第二蛋白酶切割位点的结构区；
(c)第二功能氨基酸结构区，其包含可与靶细胞表面受体结合的受体结合域和/或可以介导多肽跨囊泡膜转移的易位域。
优选的，所述的金属离子依赖的蛋白酶活性域为Zn 2+依赖的蛋白酶活性域。
优选的，所述的靶细胞为人神经细胞、胰腺细胞或其他存在SNARE复合体的细胞。
优选的，所述的单链多肽衍生物是上述单链多肽的第一蛋白酶切割位点被切割后的产物。
优选的，所述的连接短肽的长度不超过5个氨基酸。
本发明的第七方面，提供一种上述单链多肽衍生物的制备方法，所述方法包括将上述任一的单链多肽经第一蛋白酶对第一蛋白酶切割位点进行切割而获得。
本发明的第八方面，提供了一种毒素多肽的制备方法，所述制备方法包括：将上述的单链多肽的经第一蛋白酶切割去除标签蛋白，和经第二蛋白酶切割后第一功能氨基酸结构区和第二功能氨基酸结构区形成至少一个二聚体。
优选的，所述第一功能氨基酸结构区和第二功能氨基酸结构区经过二硫键连接成二聚体结构。
本发明的第九方面，提供了一种毒素多肽，所述毒素多肽包括：
(a)第一功能氨基酸结构区，其包含金属离子依赖的蛋白酶活性域；
(b)第二氨基酸结构区，其包含可与于靶细胞表面受体结合的受体结合域和/或可以介导多肽跨囊泡膜转移的易位域。
所述的(a)、(b)不通过肽键连接，优选由一个二硫键连接。
优选的，所述的毒素多肽的(a)、(b)之间形成二聚体结构。
优选的，所述的毒素多肽的(a)、(b)之间包括经第二蛋白酶切割后的氨基酸残基。
优选的，所述的毒素多肽的氨基酸序列包含如SEQ ID NO：7所示的序列。
优选的，所述的毒素多肽由上述毒素多肽的制备方法制备而获得。
优选的，相对于激活后高毒性的毒素多肽，上述毒素多肽前体(包括单链多肽或者单链多肽衍生物)具有低毒性，进一步优选的，所述低毒性是相对于激活后的高毒性而言，在一个具体的实施方式中，所述激活后的毒素多肽的活性至少高于毒素多肽前体的5000倍，更进一步，激活后的毒素多肽的活性高于毒素多肽前体的6000、7000、8000、9000、10000、12000、15000倍，甚至更高。
本发明的第十方面，提供了一种组合物，所述的组合物包含上述的经修饰的单链多肽、单链多肽衍生物或毒素多肽。
优选的，所述的组合物还包含赋形剂或稀释剂。进一步优选的，所述组合物基本不含胰蛋白酶。
优选的，所述组合物是药物组合物。
本发明的第十一方面，提供一种上述单链多肽、单链多肽衍生物或毒素多肽，在制备组合物中的用途。
优选的，所述组合物是药物组合物，所述药物组合物通过干扰神经递质释放治疗相关的疾病，优选的，所述疾病包括神经肌肉疾病、自主性障碍或疼痛，所述疾病的症状包括：痉挛性发声障碍、痉挛性斜颈、喉肌张力障碍、口下颌发音障碍、舌肌张力障碍、颈肌张力障碍、局灶性肌张力障碍、眼睑痉挛、斜视、半面痉挛、眼睑障碍、大脑性瘫痪、局灶性痉挛和其他语言障碍、痉挛性结肠炎、神经源性膀胱、盆底失迟缓、肢体痉挛、抽搐、震颤、磨牙症、肛裂、弛缓不能、吞咽困难和其他肌张力障碍以及特征为肌肉群不自主运动的其他障碍、流泪、多汗、过度流涎、过度胃肠分泌、分泌紊乱、肌肉痉挛引起的疼痛、头痛和皮肤病症。
本发明的第十二方面，提供上述的单链多肽、单链多肽衍生物、毒素多肽在通过干扰神经递质释放而改善相关症状中的非治疗用途。优选的，所述用途为美容用途，例如减少脸部皱纹，萎缩脸部肌肉达到廋脸效果等。
本发明的有益效果：
本发明提供的单链多肽，通过添加连接短肽使得标签蛋白更易于被切割，从而使得所述的单链多肽在得率更高的同时，纯度也更高。
本发明所涉及的单链多肽，其标签蛋白使得第一功能氨基酸结构区的蛋白酶结构域不能发挥蛋白酶的功能，通过将第一功能氨基酸结构区和第二功能氨基酸结构区之间的天然酶切位点替换为仅特定的酶才可以识别的位点，从而使得该单链多肽的分子活性(神经毒性)降低至天然毒素的万分之一以下，从而将生产活化分子的步骤分为了所述的单链多肽的生产和该单链多肽的活化，所述的单链多肽的活化是指让其与特定的蛋白酶接触，切开两个功能肽段之间的肽键，使其为有活性的分子。在整个生产过程中，单链多肽的生产占据了生产过程的大部分，然而由于其毒性被大幅降低，从而降低了生产过程中的危险性，使得整个工业化生产更易于进行。
本发明所述的标签蛋白，是指一种异源的亲和分子，其能够与特异的配体结合，再经过与配体的脱离，使得与标签蛋白共表达的分子更易于从不同的多肽或蛋白产物中被选择出来。
本发明生产的单链多肽，与天然来源的神经毒素相比，具有可以改变的靶位特异性结合区域或改变的活性区域或二者兼而有之。
由于得率更高、纯度更高以及安全性也更高，本发明提供的经修饰的可活化神经毒素的单链多肽相比于现有技术更易于进行生产和纯化操作，可以为临床使用而大规模制备恰当折叠和高纯度的毒素多肽。
附图说明
图1：GSTs-BoNT/A初步纯化和进一步去除GSTs标签的SDS-PAGE结果，其中第1泳道为经过GSTs亲和层析柱初步纯化得到的GSTs-BoNT/A，第2泳道为初步纯化的蛋白经过Rinovirus 3C Protease的酶切去除GSTs标签蛋白得到切除GSTs后的BoNT/A。
图2：切除了GSTs标签的产物经过离子交换柱进一步纯化得到高纯度的BoNT/A蛋白的SDS-PAGE结果。
图3：BoNT/A在还原条件下双链解离的验证结果。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
在下述实施例中，主要的设备和材料是从以下所指出的几家公司获得：
5升发酵罐(上海百伦定制)，谷胱甘肽纯化树脂(L00206)(金斯瑞生物科技股份有限公司)，3C酶(Z03092)(金斯瑞生物科技股份有限公司)。
实施例1：设计和构建编码经修饰的神经毒素的单链多肽的核酸分子：
1.核酸分子从5’端起依次包含：
(a)编码谷胱甘肽s-转移酶的核苷酸序列如SEQ ID NO：1所示，其编码的谷胱甘肽s-转移酶的氨基酸序列如SEQ ID NO：2所示；
(b)编码第一蛋白酶切割位点的核苷酸序列如SEQ ID NO：3所示，其编码的氨基酸序列如SEQ ID NO：4所示；
(c)编码GS连接短肽的核苷酸序列如SEQ ID NO：5所示，为GGATCC；
(d)编码第二多肽片段的核苷酸序列，其包含BoNT/A轻链、第二蛋白酶切割位点和BoNT/A重链的核苷酸序列，如SEQ ID NO：6所示，其编码的氨基酸序列如SEQ ID NO：7所示；其中，编码第二蛋白酶切割位点的核苷酸序列如SEQ ID NO：8所示，其编码的氨基酸序列如SEQ ID NO：9所示；
第一功能氨基酸结构区和第二功能氨基酸结构区之间也可以不存在天然的环状区域，例如去除轻链和重链之间的KTKSLDKGYNK连接序列，以减少非特异性蛋白酶切割。
编码单链多肽的核苷酸序列如SEQ ID NO：10所示，其编码的单链多肽的氨基酸序列如SEQ ID NO：11所示。
实施例2：构建包含实施例1的核酸分子的质粒
在实施例1人工合成基因工程优化后的GSTs-BoNT/A，两端合成加入NdeI和NotI酶切位点，经过NdeI和NotI 37摄氏度酶切(New England Biolabs)，使用QIquick gel extraction kit(Qiagen)纯化，使用T4 DNA ligase(NEB)插入pET28a(Novagen)质粒载体中的NdeI和NotI的位点。
实施例3：转染实施例2构建的质粒到宿主细胞
1、感受态细胞的制备：取一管大肠杆菌细胞E.coli BL21 DE3(New England Biolabs)，接种至含有3ml LB培养液的试管中，37℃振荡培养过夜.次日取菌液0.5ml接种至含有50ml LB培养基的250ml烧瓶中，37℃剧烈震荡培养约3-5小时(250rpm)，当菌落600nm OD值达到0.3-0.4时，将烧瓶取出放置冰上10-15分钟.在无菌条件下把菌液倒入50ml离心管中.4℃，1000g离心10分钟。弃上清，收集加10ml 0.1M的CaCl 2到离心管中，振荡混匀，悬浮菌体，冰浴30分钟，4℃，1000g离心10分钟，弃上清，收集加4ml冰预冷的0.1M的CaCl 2，重悬浮菌体。每管0.2ml分装，至4℃保存备用，24小时内使用，其余保存于-70℃的低温冰箱中。
2、转染：适量DNA(ng)和感受态大肠杆菌混合放置冰浴15分钟，42℃热击30秒，放置冰浴5分钟，放入SOC培养基250rpm摇晃1小时，涂抹在有抗生素的平板上，37℃隔夜培养。
实施例4：培养宿主细胞和诱导表达单链多肽
培养基的组分和配比：11.8g/L胰蛋白胨、23.6g/L酵母提取物、9.4g/L K 2HPO 4 2.2g/L KH 2PO 4 4ml/L甘油。
培养条件：37℃隔夜250rpm震荡培养细胞。
诱导表达：当大肠杆菌生长到OD 600＝1时候，加入1mM IPTG诱导表达在25℃表达5个小时。OD 600可以选用0.2-1.5，温度可以选用37℃到10℃，表达时间可以是5-16个小时。
收获细胞：3000rpm 30分钟离心收集大肠杆菌。
实施例5：单链多肽表达水平的鉴定
GSTs-BoNT/A的使用金斯瑞谷胱甘肽纯化树脂制备一根直径1厘米，高1厘米的谷胱甘肽纯化树脂层析柱，缓慢的倾倒25毫升裂解液到层析柱上层，注意不要冲起树脂，经过一小时缓慢过柱吸附裂解液中的GSTs-BoNT/A。按照常规方法洗脱获得GSTs-BoNT/A。
常规方法如下：使用20倍柱体积的磷酸缓冲液洗涤层析柱，用10倍柱体积的新鲜制备的10mM谷胱甘肽洗脱缓冲液(0.154g还原的谷胱甘肽溶解在50ml50mM Tris-HCl(pH 8.0)进行GSTs-BoNT/A洗脱，使用280nm处的吸光度读 数监控融合蛋白的洗脱。洗脱后的蛋白在Rinovirus 3C Proteas作用下切除GSTs标签蛋白。
取去除GSTs标签蛋白之前的产物和之后的产物进行常规的SDS-PAGE实验，如图1所示，与没有标签蛋白去除的第1泳道相比，在Rinovirus 3C Protease的作用下，第1泳道的GSTs标签蛋白被切除，得到没有GSTs的BoNT/A分子。
GSTs-BoNT/A在总蛋白水平中所占的比例的测定：使用4-12％SDS-PAGE(Biorad)对纯化的GSTs-BoNT/A在200伏电压下电泳分离，175kd分子量主要条带是GSTs-BoNT/A。
实施例6：标签蛋白的去除以及提纯
对将实施例5的产物进行GSTs标签蛋白的去除和有毒多肽的提纯：
用金斯瑞3C酶处理重新吸附在谷胱甘肽纯化树脂层析柱上的GSTs-BoNT/A，在3C酶的作用下，GSTs和BoNT/A之间的第一酶切位点被断开，GSTs脱离，同时BoNT/A的轻链和重链之间的第二酶切位点被断开。
以磷酸缓冲液处理谷胱甘肽纯化树脂层析柱，GSTs标签蛋白留在柱子上，被去除掉，而BoNT/A的轻链和重链被磷酸缓冲液洗脱下来。
取去除了GSTs的产物进行常规的SDS-PAGE实验，如图2所示，得到的条带中不再有GSTs标签蛋白，说明GSTs标签蛋白已经被完全去除。使用SDS-PAGE扫描图片，根据条带灰色密度来计算所得BoNT/A为90％纯度以上。
采用GSS，GSGS，GGSGS多肽代替链接短肽GS部分，其效果和GS相似，标签蛋白可以很好的暴露，从而能被完全切除。
实施例7：二聚体BoNT/A的双链在还原条件下的解离
将实施例6的产物进行还原实验：
使用100mM的二硫苏糖醇，在100摄氏度下处理样品五分钟，使样品还原，样品经过4-12％SDS-PAGE(Biorad)200伏电压电泳分离，分离出重链和轻链。如图3所示，将实施例6得到的产物在还原条件下进行还原，得到的产物进行常规的SDS-PAGE实验，得到两条不同的条带，分子量分别在100Kda和50Kda，证明实施例6形成的产物是以二硫键链接两个肽段的二聚体结构。
实施例8：GSTs-BoNT/A的毒性试验
用实施例5获得的GSTs-BoNT/A经小鼠腹腔注射的LD 50在45ng-450ng之间；考虑到所注射的肉毒素蛋白纯度，折算的LD 50在22.5ng-225ng之间，中间值为123.75ng。用实施例5获得的BoNT/A经小鼠腹腔注射药的LD 50在0.02ng-0.05ng之间。考虑到所注射的肉毒素蛋白纯度，折算的LD 50在0.006ng-0.015ng之间，中间值为0.0105ng(见表1)。
GSTs-BoNT/A具有肉毒素的活性，经小鼠腹腔注射后，其半数致死量(LD 50)大约比BoNT/A的蛋白LD 50高11786倍，表明GSTs-BoNT/A重组蛋白毒素多肽前体分子活性弱于最终产品BoNT/A大约11786倍。这个实验证明，GSTs-BoNT/A重组蛋白毒素多肽前体分子具有肉毒素的活性。由于肉毒素的超高毒性，因此生产过程中，在未对前体分子作活化处理前就需要高度安全操作防护。
表1：GSTs-BoNT/A和BoNT/A分子在小鼠生物毒性实验中的毒性比较
Figure PCTCN2022077631-appb-000001
本发明举例说明在构建包含GSTs、第一蛋白酶切割位点、连接短肽、和BoNT/A的轻链、第二蛋白酶切割位点和BoNT/A的重链的核酸分子；进一步构建包含该核酸分子的能够在E.coli中表达GSTs-BoNT/A的质粒；转染了该质粒的E.coli表达单链的GSTs-BoNT/A，该单链多肽被证明毒性是其被活化后的终产品BoNT/A毒性的万分之一以下；经过蛋白提纯和GSTs的洗脱，证明由于连接多肽的加入使得酶切位点相比没有连接短肽更易于被蛋白酶识别和切割；由于第一蛋白酶切割位点和第二蛋白酶切割位点相同，因此在GSTs被去除的同时，第二蛋白酶切割位点也被切割，第一功能氨基酸结构区和第二功能氨基酸结构区之间的肽键断开，由一个二硫键连接，形成有活性的BoNT/A，该产物被证明具有相当于天然产品的毒性。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (20)

  1. 一种经修饰的神经毒素的单链多肽，其特征在于，所述的单链多肽包含：
    (I)第一多肽片段，所述第一多肽片段包含：
    (a)标签蛋白；
    (b)包含第一蛋白酶切割位点的结构区；
    (c)连接短肽；
    (II)第二多肽片段，所述第二多肽片段包含：
    (d)第一功能氨基酸结构区，其包含金属离子依赖的蛋白酶活性域；
    (e)包含第二蛋白酶切割位点的结构区；
    (f)第二功能氨基酸结构区，其包含可与靶细胞表面受体结合的受体结合域和/或可以介导多肽跨囊泡膜转移的易位域。
  2. 如权利要求1所述的单链多肽，其特征在于，所述的第一蛋白酶切割位点和第二蛋白酶切割位点相同。
  3. 如权利要求1或2任一所述的单链多肽，其特征在于，所述的连接短肽不超过5个氨基酸残基。
  4. 如权利要求1-3任一所述的单链多肽，其特征在于，所述的第一蛋白酶切割位点和第二蛋白酶切割位点不被表达时的宿主细胞或使用时机体内源性的蛋白酶识别和切割，优选的，所述的第一蛋白酶切割位点和第二蛋白酶切割位点选自以下任一蛋白酶识别和切割的位点：非人肠激酶、烟草蚀刻病毒蛋白酶、来源于枯草芽孢杆菌的蛋白酶、来源于解淀粉芽孢杆菌的蛋白酶、来源于鼻病毒的蛋白酶、木瓜蛋白酶、昆虫木瓜蛋白酶的同源物或甲壳动物木瓜蛋白酶的同源物。
  5. 如权利要求1-4任一所述的单链多肽，其特征在于，所述的第一功能氨基酸结构区衍生自神经毒素的轻链或至少包含神经毒素的轻链的Zn 2+蛋白酶活性域；所述的第二功能氨基酸结构区衍生自神经毒素的重链或至少包含神经毒素重链的受体结合域和/或介导多肽跨囊泡膜转移的易位域。
  6. 一种编码权利要求1-5任一所述的单链多肽的核酸分子。
  7. 一种载体，其特征在于，所述的载体包含权利要求6所述的核酸分子或编码权利要求1-5任一所述的单链多肽的开放阅读框架。
  8. 如权利要求7所述的载体，其特征在于，所述的载体为质粒。
  9. 一种细胞，其特征在于，所述的细胞包含权利要求7-8任一所述的载体或表达权利要求1-5任一所述的单链多肽。
  10. 如权利要求9所述的细胞，其特征在于，所述的细胞选自任一的原核生物细胞或者真核生物细胞，优选的，所述细胞选自大肠杆菌、酵母、蓝藻或哺乳动物细胞系。
  11. 一种权利要求1-5任一所述的单链多肽的制备方法，其特征在于，所述的制备方法包括利用权利要求6所述的核酸分子，权利要求7-8任一所述的载体或者权利要求9-10任一所述的细胞制备所述的单链多肽。
  12. 一种单链多肽衍生物，其特征在于，所述的单链多肽衍生物包含：
    (I)连接短肽，以及，
    (II)第二多肽片段，所述第二多肽片段包含：
    (a)第一功能氨基酸结构区，其包含金属离子依赖的蛋白酶活性域；
    (b)包含第二蛋白酶切割位点的结构区；
    (c)第二功能氨基酸结构区，其包含可与靶细胞表面受体结合的受体结合域和/或可以介导多肽跨囊泡膜转移的易位域。
  13. 一种权利要求12所述单链多肽衍生物的制备方法，其特征在于，所述方法包括将权利要求1-5任一所述的单链多肽经第一蛋白酶对第一蛋白酶切割位点进行切割而获得。
  14. 一种毒素多肽的制备方法，其特征在于，所述制备方法包括：将权利要求1-5任一所述的单链多肽的经第一蛋白酶切割去除标签蛋白，和经第二蛋白酶切割后第一功能氨基酸结构区和第二功能氨基酸结构区形成至少一个二聚体结构。
  15. 一种毒素多肽，其特征在于，所述的毒素多肽由权利要求14的制备方法获得。
  16. 一种组合物，其特征在于，所述的组合物包含权利要求1-5任一所述的单链多肽、权利要求12所述的单链多肽衍生物或权利要求15所述的毒素多肽。
  17. 如权利要求16所述的组合物，其特征在于，所述组合物是药物组合物，优选的，所述药物组合物还包含赋形剂或稀释剂。
  18. 权利要求1-5任一所述的单链多肽、权利要求12所述的单链多肽衍生物或权利要求15所述的毒素多肽在制备组合物中的用途。
  19. 如权利要求18所述的用途，所述组合物是药物组合物，所述药物组合物通过干扰神经递质释放治疗相关的疾病，优选的，所述疾病包括神经肌肉性疾病，所述疾病的症状包括：痉挛性发声障碍、痉挛性斜颈、喉肌张力障碍、口下颌发音障碍、舌肌张力障碍、颈肌张力障碍、局灶性肌张力障碍、眼睑痉挛、斜视、半面痉挛、眼睑障碍、大脑性瘫痪、局灶性痉挛和其他语言障碍、痉挛性结肠炎、神经源性膀胱、盆底失迟缓、肢体痉挛、抽搐、震颤、磨牙症、肛裂、弛缓不能、吞咽困难和其他肌张力障碍以及特征为肌肉群不自主运动的其他障碍、流泪、多汗、过度流涎、过度胃肠分泌、分泌紊乱、肌肉痉挛引起的疼痛、头痛或皮肤病症的一种或几种。
  20. 权利要求1-5任一所述的单链多肽、权利要求12所述的单链多肽衍生物或权利要求15所述的毒素多肽在通过干扰神经递质释放而改善相关症状中的非治疗用途，优选的，所述用途为美容用途。
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