PREPARATION METHOD FOR MODIFIED TOXIN POLYPEPTIDE

Abstract

Provided is a preparation method for a modified toxin polypeptide. The preparation method comprises: step 1): expressing a modified toxin polypeptide precursor; step 2): enriching the toxin polypeptide precursor; and step 3): activating the toxin polypeptide precursor to obtain a modified toxin polypeptide.

Claims

1. A method for preparing a modified toxin polypeptide, comprising: step 1), expressing a modified toxin polypeptide precursor; step 2), enriching the toxin polypeptide precursor; and step 3), activating the toxin polypeptide precursor to obtain the modified toxin polypeptide.

2. The method according to claim 1, wherein the step 1) comprises: (1) designing a nucleic acid molecule encoding the toxin polypeptide precursor; (2) constructing a vector comprising the nucleic acid molecule; (3) transferring the nucleic acid vector into a suitable host cell; and (4) culturing the host cell, and allowing or inducing the host cell to express the toxin polypeptide precursor encoded by the nucleic acid vector.

3. The method according to claim 1, wherein the step 2) comprises enriching the toxin polypeptide precursor by conducting a multiplex filtration procedure.

4. The method according to claim 3, wherein the multiplex filtration comprises a crude liquid filtration and a feed liquid circulation filtration.

5. The method according to claim 4, wherein a material of the crude liquid filtration has a pore size of 0.1-0.65 ?m.

6. The method according to claim 4, wherein the feed liquid circulation filtration step comprises filtering a feed multiple times, and a material of the feed liquid circulation filtration has a pore size of 0.2 ?m or less.

7. The method according to claim 1, wherein the step 3) comprises digesting the toxin polypeptide precursor by using a protease to obtain the modified toxin polypeptide.

8. The method according to claim 7, wherein the toxin polypeptide precursor forms at least one dimer structure after the protease digestion.

9. The method according to claim 1, wherein the toxin polypeptide precursor has low toxicity relative to the toxin polypeptide.

10. The method according to claim 1, wherein the method further comprises step 4), purifying the toxin polypeptide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG. 1 illustrates a schematic process of the method of the present invention.

[0109] FIG. 2 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 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.

[0110] FIG. 3 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.

[0111] FIG. 4 illustrates the verification results of the dissociation of the double chains of BoNT/A under reducing conditions.

DETAILED DESCRIPTION

[0112] 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.

Example 1: Expression of GSTs-BoNT/A

[0113] The process of the method of the present invention is shown in FIG. 1, and step 1), expressing a modified toxin polypeptide, is specifically as follows:

(I): Designing and Constructing a Nucleic Acid Molecule Encoding the Modified Toxin Polypeptide Precursor:

[0114] 1. the nucleic acid molecule comprises sequentially from the 5 end: [0115] (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; [0116] (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; [0117] (c) a nucleotide sequence encoding a GS short linker peptide is set forth in SEQ ID NO: 5, which is GGATCC; [0118] (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, as set forth in SEQ ID NO: 6, the amino acid sequence encoded by the nucleotide sequence is set forth in SEQ ID NO: 7; the nucleotide sequence encoding the second protease cleavage site is set forth in SEQ ID NO: 8, and the amino acid sequence encoded by the nucleotide sequence is set forth in SEQ ID NO: 9.

[0119] 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.

[0120] The nucleotide sequence encoding a toxin polypeptide precursor is set forth in SEQ ID NO: 10, the toxin polypeptide precursor encoded by the nucleotide sequence has an amino acid sequence set forth in SEQ ID NO: 11.

(II): Constructing a Plasmid Comprising the Nucleic Acid Molecule of Step I

[0121] The genetically optimized GSTs-BoNT/A was artificially synthesized in step (I), 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).

(III): Transferring the Plasmid Constructed in Step (II) into a Host Cell

[0122] 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 transferred to an ice bath and incubated for 10-15 mM. 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 bacterial 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 at 4? C. for later use within 24 h, and the remaining samples were stored in a low-temperature freezer at ?70? C.

[0123] 2. Transfection: An appropriate amount of DNA (ng) and the competent E. coli were mixed, placed in an ice bath for 15 min, 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.

(IV): Culturing the Host Cell and Inducing the Expression of the Toxin Polypeptide Precursor

[0124] 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.

[0125] The culture condition: The cells were cultured with shaking at 250 rpm at 37? C. overnight.

[0126] Inducing expression and fermentation: The cells were transferred to a fermentor and 1 mM IPTG was added to induce the 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.

[0127] Cell lysis: The cells were sheared and degassed by conventional methods, in which the cells were lysed to obtain a cell lysate.

Example 2: Enrichment of GSTs-BoNT/A

[0128] Crude liquid filtration: The cell lysate of Example 1 was subjected to a crude liquid filtration as a crude cell lysate with a filter pore size of 0.12-0.65 ?m. The liquid that passed through the crude liquid filtration material entered the next filtration procedure as a feed liquid, and the substance that did not pass through the crude liquid filtration material entered the waste residue discharge flow path as a waste residue under the effect of a buffer.

[0129] Feed liquid filtration: The liquid after the crude liquid filtration entered a feed liquid filtration step as the feed liquid. The feed liquid was filtered multiple times (at least 2 times) in a circulation pathway, with a filter pore size of below 0.2 ?m, to obtain a finished liquid.

[0130] After the completely enclosed separation of the cell lysate, the obtained waste liquid almost contained no toxin polypeptide precursor, with very little toxicity to the operation environment, and could be directly discharged after simple disinfection treatment.

Example 3: Preliminary Purification of GSTs-BoNT/A

[0131] The finished liquid obtained in Example 2 further entered a purification module, and the GSTs-BoNT/A was obtained by a conventional affinity chromatography method.

[0132] The method is as follows: A chromatographic column was washed with 20 column volumes of a phosphate buffer. GSTs-BoNT/A was eluted with 10 column volumes of a freshly prepared 10 mM glutathione eluent buffer (0.154 g of reduced glutathione dissolved in 50 mL of 50 mM Tris-HCl (pH 8.0)). The elution of the fusion protein was monitored by absorbance reading at 280 nm.

[0133] 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 4: Activation of GSTs-BoNT/A and Dissociation of GSTs

[0134] The GSTs-BoNT/A re-adsorbed on a glutathione purification resin chromatographic column was treated using Genscript 3C enzyme. The enzyme cleavage site between GSTs and BoNT/A was cleaved under the effect of the 3C enzyme. GSTs were separated. The enzyme cleavage site between the light chain and the heavy chain of BoNT/A was also cleaved.

[0135] 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.

[0136] Products before and after the removal of the GSTs tag protein were subjected to a conventional SDS-PAGE experiment shown in FIG. 2. 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.

Example 5: Further Purification of BoNT/A

[0137] The BoNT/A obtained in Example 4 was further purified by conventional gel filtration chromatography and ion column chromatography to obtain a BoNT/A with a purity of 90% or above.

[0138] A product after the removal of GSTs was subjected to a conventional SDS-PAGE experiment shown in FIG. 3, 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.

[0139] 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 6: Western Blot Experiment

[0140] The products of Example 4 were subjected to a reduction experiment:

[0141] A sample was treated by using 100 mM dithiothreitol at 100? C. for 5 min 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.

[0142] As shown in FIG. 4, the products obtained in Example 4 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 4 had a dimeric structure in which two peptide fragments were linked by a disulfide bond.

Example 7: Toxicity Comparison of GSTs-BoNT/A and BoNT/A

[0143] The GSTs-BoNT/A obtained in Example 3 had an LD.sub.50 of 45-450 ng after intraperitoneal administration in a mice; considering the purity of the injected botulinum toxin protein, the converted LD.sub.50 was 22.5-225 ng, with a mid-value of 123.75 ng. The BoNT/A obtained in Example 4 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 LD.sub.50 was 0.006-0.015 ng, with a mid-value of 0.0105 ng (See Table 1).

TABLE-US-00001 TABLE 1 Toxicity comparison of GSTs-BoNT/A and BoNT/A molecules in mice biotoxicity study Lethality rate of mice 72 h after Test sample Description Dose (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

[0144] 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 the toxin 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 precursor molecule of the GSTs-BoNT/A recombinant protein has the activity of botulinum toxin, but toxicity much lower than that of the activated BoNT/A. Due to the ultra-high toxicity of botulinum toxin, high safety operation precautions are required in the manufacturing process even before the activation treatment of a precursor molecule.

[0145] The examples of the present application demonstrate that the method claimed in the present application is particularly suitable for the preparation of a genetically recombinant toxin polypeptide, which is activated as a toxin molecule with toxicity only by hydrolysis with a specific protease, and is expressed in the form of a mildly toxic precursor in the host cell and present in the form of the mildly toxic precursor in the cell lysate. In this case, the advantage that the enrichment of the polypeptide precursor is conducted in an enclosed system in the method avoids the tedious and costly implementation of isolation facilities, for example, an isolator, when the toxin polypeptide is industrially produced, such that the preparation method is more suitable for the large-scale industrial production of the polypeptide.

[0146] 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.

[0147] 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.