CELL LYSIS BUFFER SEPARATION APPARATUS FOR PREPARING POLYPEPTIDE, AND SYSTEM AND APPLICATION THEREOF

Abstract

A cell lysis buffer separation apparatus for preparing a polypeptide, and a system and the application thereof. The system comprises a polypeptide enrichment module, wherein the polypeptide enrichment module comprises a cell lysis buffer separation apparatus, and the cell lysis buffer separation apparatus comprises a multi-filtration device and a waste discharge pipeline, the waste discharge pipeline being connected to the multi-filtration device. The system can perform multi-filtration treatment on a lysis buffer. Therefore, the yield of target protein can be increased; and a waste liquid obtained by means of the system contains almost no toxin polypeptide precursor with low toxicity, and has very little toxicity to an operating environment, and same can be directly discharged after simple disinfection treatment, thereby greatly reducing production cost and promoting large-scale industrial production of polypeptide under the conditions of ensuring the safety of the environment and the safety of operators.

Claims

1. A cell lysis buffer separation apparatus, wherein the apparatus comprises a filtration device and a waste discharge pipeline, the filtration device is a multiplex filtration device.

2. The apparatus according to claim 1, wherein the multiplex filtration device comprises a crude liquid filtration device and a feed liquid filtration flow path, and the crude liquid filtration device and the feed liquid filtration flow path are connected via a pipeline; preferably, the feed liquid filtration flow path comprises one or more feed liquid filtration devices; more preferably, the feed liquid filtration flow path comprises a feed liquid circulation storage device and a feed liquid circulation filtration device, the feed liquid circulation storage device and the feed liquid circulation filtration device are connected end to end via a pipeline to form a feed liquid circulation filtration flow path.

3. The apparatus according to claim 1, wherein the multiplex filtration device comprises a plurality of layers of filtration devices having different filtration pore sizes; preferably, the filtration material of the crude liquid filtration device has a pore size suitable for separation of a solid and liquid in the cell lysis buffer.

4. The apparatus according to claim 3, wherein the filtration material of the multiplex filtration device is selected from hydrophilic materials or hydrophobic materials, the hydrophilic materials are selected from cellulose ester, polyethersulfone, polyethylene and the like or derivatives thereof, and the hydrophobic materials are selected from polyvinylidene fluoride, polypropylene, polytetrafluoroethylene and the like or derivatives thereof.

5. The apparatus according to claim 2, wherein the filtration material of the crude liquid filtration device has a pore size of 0.1-0.65 ?m, and the filtration material of the feed liquid circulation filtration device has a pore size of 0.2 ?m or less.

6. The apparatus according to claim 1, wherein the waste discharge pipeline comprises a waste residue discharge pipeline and a waste feed liquid discharge pipeline; preferably, the waste residue discharge pipeline is arranged downstream of the crude liquid filtration device, the waste feed liquid discharge pipeline is arranged downstream of the feed liquid filtration device.

7. The apparatus according to claim 1, wherein the apparatus further comprises a lysis buffer inlet pipeline and a supplementary liquid inlet pipeline, and the lysis buffer inlet pipeline and the supplementary liquid inlet pipeline are connected to the front end of the crude liquid filtration device; preferably, the lysis buffer inlet pipeline and buffer inlet pipeline may be the same or different.

8. The apparatus according to claim 1, wherein the apparatus further comprises a finished liquid collection flow path, the finished liquid collection flow path comprises a finished liquid collection device and a real-time turbidity monitoring device.

9. The apparatus according to claim 1, wherein the apparatus is completely enclosed.

10. A system for preparing a polypeptide, wherein the system comprises an enrichment module, the enrichment module comprises a cell lysis buffer separation apparatus, and the cell lysis buffer separation apparatus is the cell lysis buffer separation apparatus according to claim 1.

11. The system according to claim 10, wherein the system further comprises a fermentation module upstream of the enrichment module; preferably, the fermentation module comprises a culture, fermentation, and lysis device of the host cell.

12. The system according to claim 10, wherein the system further comprises a purification module downstream of the enrichment module.

13. The system according to claim 12, wherein the purification module comprises at least 1 chromatography device set; preferably, the purification module comprises 3 chromatography device sets; preferably, the chromatography device is located in an isolator; preferably, the purification module further comprises a protease liquid supply device.

14. The system according to claim 10, wherein the polypeptide is present in the form of a low-toxic single-chain polypeptide in a cell or lysis buffer and has toxicity after a downstream protease activation step; preferably, the polypeptide is a gene recombinant polypeptide.

15. The system according to claim 14, wherein the polypeptide is a neurotoxin precursor and has neurotoxicity after being activated by a protease.

16. Use of the cell lysis buffer separation apparatus according to claim 1 or the system for preparing a polypeptide in preparing a polypeptide.

17. The use according to claim 16, wherein the polypeptide is present in the form of a low-toxic single-chain polypeptide in a cell or lysis buffer and has toxicity after a downstream protease activation step; preferably, the polypeptide is a gene recombinant polypeptide.

18. The use according to claim 17, wherein the polypeptide is a neurotoxin precursor and has neurotoxicity after being activated by a protease.

19. The use according to claim 16, wherein the polypeptide comprises at least two functional amino acid structural regions, wherein a first functional amino acid structural region comprises a metal ion-dependent protease activity domain, a second functional amino acid structural region comprises a receptor-binding domain that can bind to a surface receptor of a target cell and/or a translocation domain that can mediate a transfer of the polypeptide across a vesicle membrane, and the two functional amino acid structural regions are connected via a structural region comprising a protease cleavage site.

20. The use according to claim 19, wherein the protease cleavage site is a 3C enzyme cleavage site.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0123] FIG. 1 illustrates a schematic structure of a system for preparing a polypeptide of the present invention.

[0124] FIG. 2 illustrates SDS-PAGE results of GSTs-BoNT/A (lane 1) and 150 kD BoNT/A after a GST tag had been removed (lane 2) obtained using the system of the present invention.

[0125] 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.

DETAILED DESCRIPTION

[0126] 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: System for Preparing Polypeptide

[0127] As shown in FIG. 1, a system for preparing a polypeptide sequentially comprises: [0128] (1) A fermentation module, sequentially comprising a constant temperature shaker 110, a fermentation device 120, a sealed storage tank 130, a lysis device 140, and a homogenizing device 150, wherein a buffer supply device 160 is further arranged on the sealed storage tank 130, and a waste advancing and retreating device 170 is further arranged on the lysis device 140; the above devices are connected via a pipeline, a pump 400 is arranged on the pipeline, and the pump 400 is respectively arranged between the constant temperature shaker 110 and the fermentation device 120, between the fermentation device 1:20 and the sealed storage tank 130, between the sealed storage tank 130 and the lysis device 140, between the lysis device 140 and the homogenizing device 150, and between the sealed storage tank 130 and the buffer supply device 160. [0129] (2) An enrichment module. Herein, the enrichment module refers to a cell lysis buffer processing apparatus and comprises a crude liquid filtration device 210, a feed liquid circulation storage device 221, a feed liquid circulation filtration device 222 waste residue discharge pipeline 231, a waste liquid discharge pipeline 232, a waste collection device 233, a finished liquid collection device 241, a real-time turbidity monitoring and valve device 242, and a buffer supply device 180. All the devices are connected via a pipeline, wherein the front end of the crude liquid filtration device 210 is connected with the upstream fermentation module and buffer supply device 180, the crude liquid filtration device 210 is connected with the waste residue discharge pipeline :231, and the tail end of the crude liquid filtration device 210 is connected with a feed liquid filtration device formed by connecting the feed liquid circulation storage device 221 and the feed liquid circulation filtration device 222 end to end via a pipeline; the tail end of the feed liquid circulation storage device 221 is also connected with the waste liquid discharge pipeline 232, the feed liquid circulation filtration device 22.2 is also connected with a finished liquid. collection flow path, and the finished liquid collection flow path is composed of the real-time turbidity monitoring and valve device 242 and the finished liquid collection apparatus 241. Pumps are arranged on the pipeline at the front end of the crude liquid filtration device 210 and the pipeline between the tail end of the feed liquid circulation storage device 221 and the front end of the feed liquid circulation filtration device 222. A pipeline valve is arranged on the pipeline leading from the tail end of the feed liquid circulation storage device 221 to the waste liquid discharge pipeline 232. All the devices are connected via a pipeline. The finished liquid collection device 241 is connected with a downstream purification module. The crude liquid filtration device has a pore size of 0.1-0.65 ?m, the filtration material of the feed liquid circulation filtration device has a pore size of 0.2 ?m or less, and the material may be any hydrophilic or hydrophobic filtering material.

[0130] (3) A purification module, sequentially comprising a chromatography device I 311, a chromatography device II 312, and a chromatography device III 313, wherein an activating enzyme supply device 320 is arranged at the front end of the chromatography device I 311, and a finished product collection device 330 is arranged at the tail end of the chromatography device III 313; a cleaning liquid supply device 340 and a chromatography equilibration buffer supply device 350 are arranged at the front end of each of the chromatography devices, and a waste liquid collection device 360 is arranged at the tail end of each of the chromatography devices; an isolator 370 is arranged outside each of the chromatography devices. The protease liquid supply device may further comprise two or more protease liquid supply devices, and the protease liquid comprises different proteases.

(1), (2), and (3) are connected via a pipeline.

Example 2: Expression of GSTs-BoNT/A

[0131] A host cell expressing the target polypeptide was prepared using genetic engineering technology (Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis. Cold Spring Harbor Laboratory Press: 1989), which comprises: 1. designing and constructing a nucleic acid molecule encoding a polypeptide of a modified neurotoxin; 2. constructing a plasmid containing the nucleic acid molecule in step 1; 3. transfecting the plasmid constructed in step 2 into a host cell; 4. inoculating, culturing, fermenting, lysing and homogenizing Escherichia coli expressing the polypeptide using the fermentation module in the system of the present invention.

[0132] The amino acid sequence of the expressed polypeptide is set forth in SEQ ID NO: 1.

Example 3: Enrichment of GSTs-BoNT/A

[0133] The crude cell lysis buffer obtained in Example 2 was enriched using the system in Example 1: the lysate entered the completely enclosed enrichment module via a pump, namely a completely enclosed cell lysis buffer separation and purification instrument. In the completely enclosed cell lysis buffer separation and purification instrument, firstly, a first layer of the crude liquid filtration device was used for separating the lysis buffer and residue, the residue entered a waste recycling device, and the filtered lysis buffer further entered the feed liquid circulation filtration device as a feed liquid. In the circulation filtration process, the circulation liquid meeting the finished liquid collection condition entered the finished liquid collection device, and the circulation liquid not meeting the finished liquid collection condition continued to he subjected to the circulation filtration or entered a waste liquid recycling device.

[0134] Aiming at the problems of filter membrane blockage, the need to perform liquid changing and flushing and the like in the treatment of a cell lysis buffer, in the cell lysis buffer device of the present application, in one aspect, when visible deposition occurred in the crude liquid filtration device and the passing rate was significantly reduced, the liquid in the liquid inlet pipeline was switched from the cell lysis buffer to buffer II, and the buffer II enabled the deposition in the crude liquid filtration device to be separated from the crude liquid filtration device and enter a waste residue discharge pipeline connected with the crude liquid filtration device; in another aspect, when the turbidity of the feed liquid reached a certain degree, the valve on a branch of the pipeline leading to the waste feed liquid discharge pipeline at the tail end of the feed liquid circulation storage device was opened, and the feed liquid was discharged as a waste liquid. The whole process did not need manual operations such as liquid changing, flushing and the like.

[0135] The finished liquid collection condition was that at least two circulations were performed.

[0136] By the completely enclosed cell lysis buffer separation apparatus, the obtained finished liquid contained more than 90% of polypeptide, and the obtained waste liquid almost contained single-chain polypeptide, with very little toxicity to the operating environment, and could be directly discharged after simple disinfection treatment.

Example 4: Cleavage of GSTs Tag Protein and Purification of BoNT/A

[0137] The product in Example 2 entered the purification module via the enrichment module and was contacted with a GSTs affinity ligand in a first chromatography device (an affinity chromatography device). Substances which were not bound to the ligand were eluted by a conventional method, and the GSTs were dissociated from the ligand by a conventional method to obtain the preliminarily purified GSTs-BoNT/A.

[0138] The conventional method is as follows: a chromatographic column was washed with 20 column volumes of a phosphate buffer. GSTs-BoNT/A elution was carried out 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.

[0139] After elution, GSTs tag protein was cleaved under the effect of Rinovirus 3C Protease.

[0140] As 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 oaf Rinovirus 3C Protease.

[0141] The preliminarily purified product was further purified in a second chromatography device (a gel filtration chromatography device), or a third chromatography device (an ion chromatography device). The purification method was well known to those skilled in the art. The results are shown in FIG. 3. The band corresponding to the polypeptide is significantly thickened, and a BoNT/A molecule with the purity of 90% can be obtained.

Example 5: Toxicity Test of GSTs-BoNT/A and BoNT/A

[0142] The GSTs-BoNT/A obtained in Example 4 had an LD.sub.50 of 45-450 ng after intraperitoneal injection in 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 injection 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

[0143] 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 injection 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. Therefore, the cell lysis buffer separation apparatus in which the cell lysis butter contains the the toxin polypeptide precursor is subjected to a completely enclosed treatment, namely the blocking function included in the apparatus pipeline, such as a rubber gasket, a bolt, and a stainless steel interface, and in the operation process, the apparatus does not need to be disconnected to carry out manual operations such as flushing, liquid changing and the like. The completely enclosed cell lysis buffer processing apparatus can completely process the cell lysis buffer containing the low-toxic toxin polypeptide precursor without an additional enclosed working space, such as an isolator.

[0144] The examples of the present application demonstrate that the system for preparing a polypeptide claimed in the present application can be applied to the preparation of a toxic polypeptide, which is activated as a toxin molecule with natural toxicity only by hydrolysis with a specific protease, and is present in a low-toxic form in the host cell and cell lysis buffer. In this case, cell fermentation, lysis, and lysis buffer separation can all be performed in the enclosed system of the present application without the need for an isolator in these steps, which in one aspect improves the safety of the production of low-toxic polypeptides and in another aspect reduces the production cost. Meanwhile, the multi-step chromatography purification after the protease activation further improves the purity of the active polypeptide, and the purity can reach 90%, thereby enabling the large-scale industrial production of such polypeptides to be possible.

[0145] 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.

[0146] 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.