MODIFIED NEUROTOXIN SINGLE-CHAIN POLYPEPTIDE AND USE THEREOF

Abstract

The present invention relates to a modified neurotoxin single-chain polypeptide and a use thereof. The single-chain polypeptide contains a tagged protein, a linker short peptide, etc. The linker short peptide facilitates cleavage of the tagged protein, thereby improving purification of the single-chain polypeptide; in addition, the single-chain polypeptide has neurotoxicity when activated, but the single-chain polypeptide per se is only slightly toxic, such that the production safety is improved and the production process cost is reduced while the yield of the single-chain polypeptide is greatly increased.

Claims

1. A modified single-chain polypeptide of a neurotoxin, comprising: (I) a first polypeptide fragment, comprising: (a) a tag protein, (b) a structural region comprising a first protease cleavage site, (c) a short linker peptide; (II) a second polypeptide fragment, comprising: (d) a first functional amino acid structural region, comprising a metal ion-dependent protease activity domain; (e) a structural region comprising a second protease cleavage site; (f) a second functional amino acid structural region, comprising a receptor-binding domain that can bind to a surface receptor of a target cell and/or a translocation domain that can mediate the transfer of the polypeptide across the vesicle membrane.

2. The single-chain polypeptide according to claim 1, wherein the first protease cleavage site and the second protease cleavage site are identical.

3. The single-chain polypeptide according to claim 1, wherein the short linker peptide has no more than 5 amino acid residues.

4. The single-chain polypeptide according to claim 1, wherein the first protease cleavage site and the second protease cleavage site are not recognized and cleaved by a host cell when expressed or by an endogenous protease of a body when used; preferably, the first protease cleavage site and the second protease cleavage site are selected from any one of the following protease recognition and cleavage sites: a non-human enterokinase, a tobacco etch virus protease, a protease derived from Bacillus subtilis, a protease derived from Bacillus amyloliquefaciens, a protease derived from rhinovirus, a papain, a homologue of papain of an insect, or a homologue of papain of a crustacean.

5. The single-chain polypeptide according to claim 1, wherein the first functional amino acid structural region is derived from a light chain of the neurotoxin or comprises at least a Zn.sup.2+ protease activity domain of the light chain of the neurotoxin; the second functional amino acid structural region is derived from a heavy chain of the neurotoxin or comprises at least a receptor-binding domain of the heavy chain of the neurotoxin and/or a translocation domain mediating a transfer of the polypeptide across a vesicle membrane.

6. A nucleic acid molecule encoding the single-chain polypeptide according to claim 1.

7. A vector, comprising the nucleic acid molecule according to claim 6 or an open reading frame encoding the single-chain polypeptide.

8. The vector according to claim 7, wherein the vector is a plasmid.

9. A cell, comprising the vector according claim 7 or expressing the single-chain polypeptide.

10. The cell according to claim 9, wherein the cell is selected from any prokaryotic or eukaryotic cell; preferably, the cell is selected from Escherichia coli, yeast, cyanobacteria or mammalian cell lines.

11. A method for preparing the single-chain polypeptide according to claim 1, comprising preparing the single-chain polypeptide using the nucleic acid molecule, the vector according to or the cell.

12. A single-chain polypeptide derivative, comprising: (I) a short linker peptide; and (II) a second polypeptide fragment, comprising: (a) a first functional amino acid structural region, comprising a metal ion-dependent protease activity domain; (b) a structural region comprising a second protease cleavage site; (c) a second functional amino acid structural region, comprising a receptor-binding domain that can bind to a surface receptor of a target cell and/or a translocation domain that can mediate the transfer of the polypeptide across the vesicle membrane.

13. A method for preparing the single-chain polypeptide derivative according to claim 12, comprising cleaving the single-chain polypeptide at the first protease cleavage site with the first protease to obtain the single-chain polypeptide derivative.

14. A method for preparing a toxin polypeptide, comprising cleaving the single-chain polypeptide according to claim 1 with the first protease to remove the tag protein, and forming at least one dimeric structure with the first functional amino acid structural region and the second functional amino acid structural region after cleaving with the second protease.

15. A toxin polypeptide prepared by the method according to claim 14.

16. A composition, comprising the single-chain polypeptide according to claim 1, the single-chain polypeptide derivative or the toxin polypeptide.

17. The composition according to claim 16, wherein the composition is a pharmaceutical composition; preferably, the pharmaceutical composition further comprises an excipient or diluent.

18. Use of the single-chain polypeptide according to claim 1, the single-chain polypeptide derivative or the toxin polypeptide in preparing a composition.

19. The use according to claim 18, wherein the composition is a pharmaceutical composition for treating a related disease by interfering with neurotransmitter release; preferably, the disease includes a neuromuscular disease, symptoms of the disease include one or more of spasmodic vocalization disorder, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, tongue dystonia, cervical dystonia, focal dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasm and other language disorders, spastic colitis, neurogenic bladder, anismus, limb spasm, tics, tremor, bruxism, anal fissure, achalasia, dysphagia and other dystonias as well as other disorders characterized by muscle group involuntary movement, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, secretory disorders, pain due to muscle spasm, headache or a skin disorder.

20. Non-therapeutic use of the single-chain polypeptide according to claim 1, the single-chain polypeptide derivative or the toxin polypeptide in ameliorating a related condition by interfering with neurotransmitter release, wherein the use is preferably a cosmetic use.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0107] FIG. 1 illustrates the SDS-PAGE results of the preliminary purification of GSTs-BoNT/A and the further removal of GSTs tags, wherein lane 1 is the GSTs-BoNT/A obtained in the preliminary purification by a GSTs affinity chromatographic column, and lane 2 is BoNT/A without GSTs obtained by removing the GSTs tag protein through the digestion of the preliminarily purified protein with Rinovirus 3C Protease.

[0108] FIG. 2 illustrates the SDS-PAGE results of a high-purity BoNT/A protein obtained by further purifying the product without GSTs tag through an ion exchange column.

[0109] FIG. 3 illustrates the verification results of the dissociation of double chains of BoNT/A under reducing conditions.

DETAILED DESCRIPTION

[0110] The present invention is further described with reference to the following specific examples, and the advantages and features of the present invention will be clearer as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It should be appreciated by those skilled in the art that modifications and replacements can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention and that all these modifications and replacements fall within the scope of the present invention.

[0111] In the following examples, the main equipment and materials were obtained from several companies indicated below:

[0112] 5-liter fermenter (customized by Shanghai Bailun), glutathione purified resin (L00206) (Genscript Biotechnology Co., Ltd.), and 3C enzyme (Z03092) (Genscript Biotechnology Co., Ltd.).

Example 1: Design and Construction of Nucleic Acid Molecule Encoding Modified Single-Chain Polypeptide of Neurotoxin

[0113] 1. the nucleic acid molecule comprises sequentially from the 5 end: [0114] (a) a nucleotide sequence encoding a glutathione S-transferase is set forth in SEQ ID NO: 1, the glutathione S-transferase encoded by the nucleotide sequence has an amino acid sequence set forth in SEQ ID NO: 2; [0115] (b) a nucleotide sequence encoding a first protease cleavage site is set forth in SEQ ID NO: 3, the amino acid sequence encoded by the nucleotide sequence is set forth in SEQ ID NO: 4; [0116] (c) a nucleotide sequence encoding a GS short linker peptide is set forth in SEQ ID NO: 5, which is GGATCC; [0117] (d) a nucleotide sequence encoding a second polypeptide fragment comprising nucleotide sequences of the light chain of BoNT/A, a second protease cleavage site, and the heavy chain of BoNT/A, is set forth in SEQ ID NO: 6, the amino acid sequence encoded by the nucleotide sequence is set forth in SEQ ID NO: 7; wherein the nucleotide sequence encoding the second protease cleavage site is set forth in SEQ ID NO: 8, the amino acid sequence encoded by the nucleotide sequence is set forth in SEQ ID NO: 9.

[0118] There can also be no natural loop region between the first functional amino acid structural region and the second functional amino acid structural region. For example, the KTKSLDKGYNK linker sequence between the light chain and the heavy chain may be removed to reduce non-specific protease cleavage.

[0119] The nucleotide sequence encoding a single-chain polypeptide is set forth in SEQ ID NO: 10, the single-chain polypeptide encoded by the nucleotide sequence has an amino acid sequence set forth in SEQ ID NO: 11.

Example 2: Construction of Plasmid Comprising Nucleic Acid Molecule in Example 1

[0120] The genetically optimized GSTs-BoNT/A was artificially synthesized in Example 1, and NdeI and NotI enzyme cleavage sites were synthesized and added at the two ends thereof. The GSTs-BoNT/A was digested with the NdeI and NotI at 37? C. (New England Biolabs), purified by using a QIquick gel extraction kit (Qiagen), and inserted into NdeI and NotI sites in a pET28a (Novagen) plasmid vector by using a T4 DNA ligase (NEB).

Example 3: Transfection of Plasmid Constructed in Example 2 into Host Cell

[0121] 1. Preparation of competent cells: A tube of E.coli BL21 DE3 cells (New England Biolabs) was inoculated into a test tube containing 3 mL of LB culture medium and cultured with shaking at 37? C. overnight. On the next day, 0.5 mL of the bacterial culture was inoculated into a 250-mL flask containing 50 mL of LB culture medium and cultured with vigorous shaking (250 rpm) at 37? C. for about 3-5 h. When the OD value of the bacterial colony reached 0.3-0.4 at 600 nm, the flask was taken out and placed on ice for 10-15 min. The bacterial culture was poured into a 50 mL centrifuge tube and centrifuged at 1000 g at 4? C. for 10 mM under an aseptic condition. The supernatant was discarded, and the cells were collected. 10 mL of 0.1 M CaCl.sub.2 was added into the centrifuge tube, and the mixture was mixed well with shaking to resuspend the bacteria cells. The cells were then subjected to an ice bath for 30 mM, and centrifuged at 1000 g at 4? C. for 10 mM. The supernatant was discarded, and 4 mL of 0.1 M CaCl.sub.2 pre-cooled with ice was added to resuspend the collected bacterial cells. The cells were aliquoted at 0.2 mL per tube and stored for later use within 24 h, and the remaining samples were stored in a low-temperature freezer at ?70? C.

[0122] 2. Transfection: An appropriate amount of DNA (ng) and the competent E. coli were mixed, placed in an ice bath for 15 mM, heat-shocked at 42? C. for 30 s, placed in the ice bath for 5 mM, shaken at 250 rpm for 1 h in an SOC medium, smeared on a plate with antibiotics, and cultured at 37? C. overnight.

Example 4: Culture of Host Cell and Induction of Expression of Single-Chain Polypeptide

[0123] The composition and ratio of the culture medium: 11.8 g/L of tryptone, 23.6 g/L of yeast extract, 9.4 g/L of K.sub.2HPO.sub.4, 2.2 g/L of KH.sub.2PO.sub.4, and 4 mL/L of glycerol.

[0124] The culture condition: The cells were cultured with shaking at 250 rpm at 37? C. overnight.

[0125] Inducing expression: 1 mM IPTG was added to induce expression at 25? C. for 5 h when the E. coli grew to a stage in which the OD.sub.600=1. The OD.sub.600 threshold may be 0.2-1.5, the temperature threshold may be 37? C. to 10? C., and the expression time may be 5-16 h. Harvesting cells: E. coli was collected by centrifugation at 3000 rpm for 30 mM.

Example 5: Identification of Expression Level of Single-Chain Polypeptide

[0126] GSTs-BoNT/A: A glutathione purification resin chromatographic column having a diameter of 1 cm and a height of 1 cm was prepared using Genscript glutathione purification resin, 25 mL of the lysate was slowly poured to the upper layer of the chromatographic column (be careful not to flush the resin), and the lysate was slowly loaded on the column to adsorb GSTs-BoNT/A in the lysate for one hour. The GSTs-BoNT/A was eluted according to a conventional method.

[0127] The conventional method is as follows: A chromatographic column was washed with 20 column volumes of a phosphate buffer. GSTs-BoNT/A elution was eluted with 10 column volumes of a freshly prepared 10 mM glutathione elution buffer (0.154 g reduced glutathione dissolved in 50 mL of 50 mM Tris-HC1 (pH 8.0)). The elution of the fusion protein was monitored by absorbance reading at 280 nm. GSTs tag protein of the eluted protein was cleaved under the effect of a Rinovirus 3C Protease.

[0128] Products before and after the removal of the GSTs tag protein were subjected to a conventional SDS-PAGE experiment shown in FIG. 1. Compared with lane 1 in which the tag protein was not cleaved, the GSTs tag protein of lane 1 was cleaved to obtain a BoNT/A molecule without GSTs under the effect of Rinovirus 3C Protease.

[0129] Determination of the proportion of GSTs-BoNT/A in the total protein level: The purified GSTs-BoNT/A was electrophoretically separated at 200 volts by using 4-12% SDS-PAGE (Biorad) and a major band with a molecular weight of 175 kd was GSTs-BoNT/A.

Example 6: Removal of Tag Protein and Purification

[0130] The product in Example 5 was subjected to removal of GSTs tag protein and purification of toxic polypeptides:

[0131] The GSTs-BoNT/A re-adsorbed on a glutathione purification resin chromatographic column was treated using Genscript 3C enzyme. The first enzyme cleavage site between the GSTs and BoNT/A was cleaved under the effect of the 3C enzyme. GSTs were separated. The second enzyme cleavage site between the light chain and the heavy chain of BoNT/A was cleaved.

[0132] The glutathione purification resin chromatographic column was treated with a phosphate buffer. The GSTs tag protein was retained on the column and was thus removed, while the light chain and the heavy chain of BoNT/A were eluted by the phosphate buffer.

[0133] A product after the removal of GSTs was subjected to a conventional SDS-PAGE experiment shown in FIG. 2, and the obtained bands did not include the GSTs tag protein, indicating that the GSTs tag protein had been completely removed. The SDS-PAGE results were scanned, and the purity of the obtained BoNT/A was above 90% by calculation based on the grey density of the band.

[0134] GSS, GSGS and GGSGS polypeptides were adopted to replace a GS part of a short linker peptide, which demonstrated a similar effect to that of GS. The tag protein can be well exposed and thus completely cleaved.

Example 7: Dissociation of Dimer BoNT/A Double Chains under Reducing Conditions

[0135] The products of Example 6 were subjected to a reduction experiment:

[0136] A sample was treated by using 100 mM dithiothreitol at 100? C. for 5 mM to reduce the sample, and electrophoretically separated at 200 volts by the 4-12% SDS-PAGE (Biorad) to separate the heavy chain and the light chain. As shown in FIG. 3, the products obtained in Example 6 were subjected to reduction under reducing conditions, and the obtained products were subjected to a conventional SDS-PAGE experiment to obtain two different bands with molecular weights of 100 KDa and 50 KDa, respectively, which demonstrated that the product formed in Example 6 was a dimeric structure in which two peptide fragments were linked by a disulfide bond.

Example 8: Toxicity Test of GSTs-BoNT/A

[0137] The GSTs-BoNT/A obtained in Example 5 had an LDso of 45-450 ng after intraperitoneal administration in mice; considering the purity of the injected botulinum toxin protein, the converted LDso was 22.5-225 ng, with a mid-value of 123.75 ng. The BoNT/A obtained in Example 5 had an LD.sub.50 of 0.02-0.05 ng after intraperitoneal administration in mice. Considering the purity of the injected botulinum toxin protein, the converted LDso was 0.006-0.015 ng, with a mid-value of 0.0105 ng (see Table 1).

[0138] The GSTs-BoNT/A had the activity of botulinum toxin, and the median lethal dose (LD.sub.50) thereof was approximately 11786 times higher than the LD.sub.50 of the BoNT/A protein after intraperitoneal administration in mice, which indicates that the activity of a toxin polypeptide precursor molecule of GSTs-BoNT/A recombinant protein is approximately 11786 times weaker than that of the final product BoNT/A. The experiment demonstrates that the toxin polypeptide precursor molecule of the GSTs-BoNT/A recombinant protein has the activity of botulinum toxin. Due to the ultra-high toxicity of botulinum toxin, high safety operation precautions are required in the manufacturing process before the activation treatment of a precursor molecule.

TABLE-US-00001 TABLE 1 comparison of GSTs-BoNT/A and BoNT/A molecules in mice biotoxicity study Lethality rate of Dose mice 72 h after Test sample Description (ng) administration (%) GST-BoNT/A GST-BoNT/A 450 100 (50% purity) (50% purity) 45 50 4.5 0 0.45 0 BoNT/A BoNT/A 0.05 100 (30% purity) (30% purity) 0.02 0 0.01 0 0.005 0

[0139] The present invention exemplifies the construction of a nucleic acid molecule comprising GSTs, a first protease cleavage site, a short linker peptide, and a light chain of BoNT/A, a second protease cleavage site, and a heavy chain of BoNT/A; further constructs a plasmid containing the nucleic acid molecule and capable of expressing GSTs-BoNT/A in E.coli; E.coli transfected with the plasmid expresses single-chain GSTs-BoNT/A, the single-chain polypeptide is proved to be less than ten thousandth of toxicity of the activated final product BoNT/A. Protein purification and GSTs elution prove that due to the addition of the short linker peptide, enzyme cleavage sites are easier to identify and cleave by a protease than the enzyme cleavage sites without a short linker peptide. Since the first protease cleavage site and the second protease cleavage site are identical, when GSTs are removed, the second protease cleavage site is cleaved, and the peptide bond between the first functional amino acid structural region and the second functional amino acid structural region is cleaved and the two structural regions are linked by a disulfide bond to form active BoNT/A, which is proved to have toxicity equivalent to that of a natural product.

[0140] The preferred embodiments of the present invention are described in detail above, which, however, are not intended to limit the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, all of which will fall within the protection scope of the present invention.

[0141] In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner where the features do not contradict each other. In order to avoid unnecessary repetition, such combinations will not be illustrated separately.