A system for preparing polypeptides and uses thereof

Abstract

The present application relates to a kind of preparation of polypeptide system and its application, the system includes polypeptide enrichment module, the polypeptide enrichment module includes cell lysate filtration equipment, the cell lysate filtration equipment includes multiple filtration device, waste discharge pipeline, the waste discharge pipeline is connected with multiple filtration device.The system can carry out multiple filtration treatment to lysate, on the one hand, the yield of target protein can be improved, on the other hand, the waste liquid obtained by the system is almost free of low-toxicity toxin polypeptide precursor, the toxicity to operating environment is very small, can be directly discharged after simple disinfection, so as to ensure the safety of environment, the safety of operator, greatly reduce production cost, promote the large-scale industrial production of polypeptide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and manufacturing, and particularly relates to a system for preparing polypeptides and application thereof. BACKGROUND

[0002] Polypeptides are a class of compounds formed by connecting multiple amino acids through peptide bonds, usually composed of 10-100 amino acid molecules, with the same connection mode as proteins and a relative molecular mass lower than 10000. Polypeptides are widely present in living organisms, and to date, tens of thousands of polypeptides have been found in living organisms, which widely participate in and regulate the functional activities of systems, organs, tissues and cells in the body and play an important role in life activities.

[0003] Polypeptide drugs refer to polypeptides with specific therapeutic effects extracted from animals and plants or chemically synthesized or genetically recombined, which are specific applications of polypeptides in the medical field. Although the development of polypeptide drugs is relatively short, the development is very rapid, and polypeptide drugs have become a hot spot in market development. Polypeptide drugs are mainly used for treating cancer and major diseases related to metabolic disorders, and the drugs related to these diseases have a very important market worldwide. At present, 200-300 polypeptide drugs are in clinical trials, 500-600 are in preclinical trials, and more polypeptide drugs are in the laboratory research stage.

[0004] With the maturity of biological recombination technology, using prokaryotes or eukaryotes as host cells to mass-produce recombinant polypeptide molecules, especially long-chain polypeptides, is an important way to produce recombinant polypeptides. Recombinant polypeptide technology has brought great leap in the production of enzyme preparations and polypeptide drugs, but the price of polypeptide biological products (especially polypeptide drugs) is still high. An important reason is that the synthesis and separation and purification process of recombinant polypeptides are complex, and the production and purification cost is high. Therefore, how to realize the efficient and large-scale expression of recombinant polypeptides, and simplify the separation process and reduce the separation cost, is an important research topic in industrial biotechnology.

[0005] Especially for the preparation of some toxin polypeptides, due to the dual requirements of protecting the environment and the operating personnel, some large-volume material processing needs to be carried out in a BL-3 environment or in a large-scale isolator, which undoubtedly increases the production cost and is not conducive to large-scale industrial production. For example, the existing botulinum toxin industrial preparation system still needs to be carried out in a BL-3 environment, and all cell culture and processing of large-volume cell lysate are carried out in a large-scale isolator, which faces great challenges in production, and this preparation system still has a great risk of environmental pollution. SUMMARY

[0006] In order to overcome the above-mentioned defects, the present application provides an improved system for preparing polypeptides and its application. According to the controllable toxicity of toxin polypeptides in the production process, a separation protection device is designed to adapt to the toxicity level of each stage. On the one hand, the production cost is greatly reduced, and on the other hand, the environment is strictly protected. Specifically,

[0007] In the first aspect of the present application, a system for preparing polypeptides is provided, which comprises an enrichment module, wherein the enrichment module comprises a cell lysate separation device, and the cell lysate separation device comprises a multiple filtration device and a waste discharge pipeline connected with the multiple filtration device.

[0008] Preferably, the cell lysate separation device is fully enclosed.

[0009] Preferably, the multiple filtration device comprises a crude liquid filtration device and a feed liquid filtration flow path.

[0010] Preferably, the crude liquid refers to the cell lysate before filtration. The feed liquid refers to the material after filtration by the crude liquid filtration device.

[0011] Preferably, the waste discharge pipeline comprises a waste residue discharge pipeline and a waste liquid discharge pipeline.

[0012] Preferably, the waste residue discharge pipeline is arranged on the crude liquid filtration device. The waste liquid discharge pipeline is arranged on the feed liquid filtration flow path.

[0013] Preferably, the waste residue is a material that cannot pass through the crude liquid filtration device. The waste residue enters the waste residue discharge pipeline for discharge.

[0014] Preferably, the front end of the crude liquid filtration device is connected with a lysate inlet pipeline and a buffer solution inlet pipeline. The lysate inlet pipeline and the buffer solution inlet pipeline can be the same or different. In a specific embodiment, the lysate inlet pipeline and the buffer solution inlet pipeline are the same.

[0015] Preferably, the buffer solution is used to flush the waste residue deposited on the crude liquid filtration device.

[0016] Preferably, the feed liquid filtration flow path comprises one or more feed liquid filtration devices.

[0017] Preferably, the feed liquid filtration device can be recycled.

[0018] Preferably, the feed liquid filtration device comprises a feed liquid circulation filtration device and a feed liquid circulation storage device.

[0019] Preferably, the feed liquid circulation filtering device and the feed liquid circulation storage device form a loop, i.e. the end of the feed liquid circulation storage device is connected to the feed liquid circulation filtering device through a pipeline, and the end of the feed liquid circulation filtering device is connected to the front end of the feed liquid circulation storage device through a pipeline.

[0020] Preferably, the waste liquid discharge pipeline is arranged on the feed liquid filtering flow path.

[0021] Preferably, a pipeline valve is arranged between the waste liquid discharge pipeline and the feed liquid filtering flow path.

[0022] Preferably, the waste liquid discharge pipeline is arranged at the end of the feed liquid circulation storage device.

[0023] In a specific embodiment, the waste liquid discharge pipeline is arranged on the branch pipeline of the end pipeline of the feed liquid circulation storage device.

[0024] Preferably, a pipeline valve is arranged between the waste liquid discharge pipeline and the branch pipeline of the end pipeline of the feed liquid circulation storage device.

[0025] Preferably, the cell lysate separation device further comprises a product liquid collection flow path.

[0026] Preferably, the product liquid collection flow path is connected to the feed liquid filtering flow path.

[0027] Preferably, the product liquid collection flow path is connected to the feed liquid circulation filtering device through a pipeline, and the product liquid collection flow path comprises a product liquid collection device and a real-time turbidity monitoring device.

[0028] Preferably, a valve is arranged on the pipeline between the product liquid collection device and the feed liquid circulation filtering device.

[0029] Preferably, the real-time turbidity monitoring device controls the valve.

[0030] Preferably, a pipeline valve is arranged between the crude liquid filtering device and the feed liquid filtering flow path.

[0031] Preferably, a pump is arranged on the lysate inlet pipeline.

[0032] Preferably, a pump is arranged on the supplement liquid inlet pipeline.

[0033] Preferably, a pump is arranged on the pipeline between the end of the feed liquid circulation storage device and the front end of the feed liquid circulation filtering device.

[0034] Preferably, the materials and pore sizes of each pipeline are the same or different.

[0035] Preferably, the valves are the same or different.

[0036] Preferably, the pumps are the same or different.

[0037] Preferably, the multiple filtration device comprises multiple layers of filtration devices with different pore sizes.

[0038] Preferably, the pore size of the filtration material of the crude liquid filtration device is suitable for separating the solid and liquid in the cell lysate.

[0039] Preferably, the pore size of the filtration material of the crude liquid filtration device is 0.1-0.65 μm.

[0040] Preferably, the pore size of the filtration material in the feed liquid circulation filtration device is below 0.2 μm.

[0041] Preferably, the material in the lysate that can pass through the crude liquid filtration device is circulated into the feed liquid circulation pipeline as the feed liquid.

[0042] Preferably, the feed liquid circulation that still cannot reach the clear degree of the finished liquid after a certain number of circulation filtrations is circulated into the feed liquid circulation pipeline as the waste liquid.

[0043] Preferably, the filtration material is selected from hydrophilic materials or hydrophobic materials.

[0044] Preferably, the hydrophilic filtration material is selected from cellulose ester, polyether sulfone, etc. or their derivatives.

[0045] Preferably, the hydrophobic filtration material is selected from polyvinylidene fluoride, polypropylene, polyethylene, polytetrafluoroethylene, etc. or their derivatives.

[0046] Preferably, the system further comprises a fermentation module, and the fermentation module is located upstream of the enrichment module.

[0047] Preferably, the fermentation module comprises a host cell culture device and a lysis device, and the lysis device is arranged downstream of the culture device.

[0048] Preferably, the fermentation module further comprises a sealed storage device for the host cell fermentation liquid, and the sealed storage device is arranged between the culture device and the lysis device.

[0049] Preferably, the fermentation module further comprises a homogenization device, and the homogenization device is arranged downstream of the lysis device.

[0050] Preferably, the sealed storage device is further provided with a buffer supply device I.

[0051] Preferably, the lysis device is further provided with a waste feed and discharge device.

[0052] Preferably, the system further comprises a purification module, which is located downstream of the enrichment module.

[0053] Preferably, the purification module comprises at least one set of chromatography device.

[0054] Preferably, the chromatography device comprises two or three sets of chromatography device.

[0055] Preferably, the chromatography device is the same or different, more preferably, the chromatography device is affinity chromatography device, gel filtration chromatography device and / or ion chromatography device.

[0056] Preferably, the chromatography device is located in a separator.

[0057] Preferably, the purification module further comprises a protease solution supply device.

[0058] Preferably, the protease solution supply device is connected to the front end of the first chromatography device.

[0059] Preferably, the protease solution supply device comprises two or more, and the protease solution comprises different proteases.

[0060] Preferably, the purification module comprises a product collection device, which is arranged at the end of the last chromatography device.

[0061] Preferably, the purification module further comprises a washing solution supply device, which is arranged upstream of each chromatography device.

[0062] Preferably, the purification module further comprises a balancing solution supply device, which is arranged upstream of each chromatography device.

[0063] Preferably, the balancing solution supply device is arranged upstream of the second and third chromatography devices.

[0064] Preferably, the purification module further comprises a waste liquid collection device, which is arranged below each chromatography device.

[0065] Preferably, the polypeptide exists in the form of low-toxicity single-chain polypeptide in the cell or lysate, and has high toxicity after the downstream protease (also known as activation enzyme) activation step.

[0066] Further preferably, the low toxicity is relative to the high toxicity after activation, and in a specific embodiment, the activity of the activated toxin polypeptide is at least 5000 times higher than that of the single-chain polypeptide, and further, the activity of the activated toxin polypeptide is 6000, 7000, 8000, 9000, 10000, 12000, 15000 times or even higher than that of the single-chain polypeptide.

[0067] Preferably, the polypeptide is a genetically recombined polypeptide.

[0068] Preferably, the single chain polypeptide is a toxin polypeptide precursor.

[0069] In a specific embodiment, the toxin polypeptide precursor is a neurotoxin precursor which, upon activation by a protease, i.e. an activator enzyme, has neurotoxicity.

[0070] In a second aspect, the present application provides a use of the above system in the preparation of a polypeptide.

[0071] Preferably, the polypeptide is a single chain polypeptide which is present in the cell or lysate in a non-toxic form and which, upon a downstream protease activation step, has toxicity. Preferably, the polypeptide is a genetically recombined polypeptide.

[0072] Preferably, the single chain polypeptide is a toxin polypeptide precursor.

[0073] In a specific embodiment, the toxin polypeptide precursor is a neurotoxin precursor which, upon activation by a protease, i.e. an activator enzyme, has neurotoxicity.

[0074] Preferably, the polypeptide comprises at least two functional amino acid domains, wherein the first functional amino acid domain comprises a metal ion-dependent protease active domain, the second functional amino acid domain comprises a receptor binding domain which can bind to a target cell surface receptor and / or a translocation domain which can mediate the translocation of the polypeptide across a vesicle membrane, and the two functional amino acid domains are connected by a domain comprising a protease cleavage site.

[0075] Preferably, the functional amino acid domains are derived from or are derived from the same or different natural polypeptides.

[0076] Preferably, the functional amino acid domains are computer designed.

[0077] Preferably, the domain comprising the two functional amino acid domains and the protease cleavage site (the second protease cleavage site) is defined as a second polypeptide fragment.

[0078] Preferably, the metal ion-dependent protease active domain is a Zn 2+ dependent protease active domain.

[0079] Preferably, the first functional amino acid domain and / or the second functional amino acid domain is encoded by a natural sequence and / or a synthetic sequence. More preferably, the first functional amino acid domain of the polypeptide comprises a Zn 2+ protease binding domain of a clostridial toxin light chain.

[0080] Preferably, the human cell to which the receptor binding domain of the second functional amino acid domain is capable of binding is a human cell that has a SNARE complex. For example, a human neuronal cell, a pancreatic cell or any other cell that has a SNARE complex.

[0081] More preferably, the human cell to which the receptor binding domain of the second functional amino acid domain is capable of binding is a human neuronal cell or a pancreatic cell, and the receptor binding domain is a receptor binding domain that specifically binds to a human neuronal cell or a pancreatic cell.

[0082] More preferably, the receptor binding domain of the second functional amino acid domain is a cell surface receptor binding domain of a heavy chain of a Clostridial toxin, and the translocation domain of the second functional amino acid domain that mediates the translocation of the polypeptide across a membrane is a domain that mediates the translocation of a Clostridial toxin across a membrane.

[0083] More preferably, the Clostridial toxin is a botulinum toxin or a tetanus toxin.

[0084] More preferably, the botulinum toxin is selected from any of the serotypes BoNT / A-BoNT / H and derivatives thereof known in the art.

[0085] More preferably, the Clostridial toxin is selected from any of tetanus toxin or derivatives thereof. More preferably, the first functional amino acid domain comprises part or all of the light chain of BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / H or tetanus toxin.

[0086] More preferably, the second functional amino acid domain comprises part or all of the heavy chain of BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / H or tetanus toxin.

[0087] In one embodiment, the first functional amino acid domain is the light chain of BoNT / A and the second functional amino acid domain is the heavy chain of BoNT / A.

[0088] More preferably, the first and second functional amino acid domains can be from different serotypes. Any combination of the above serotypes can be used, for example, the first functional amino acid domain can be derived from the light chain of BoNT / A and the second functional amino acid domain can be derived from the heavy chain of BoNT / B, the first functional amino acid domain can be derived from the light chain of BoNT / A and the second functional amino acid domain can be derived from the heavy chain of BoNT / C, and so on.

[0089] Preferably, the polypeptide further comprises a first polypeptide segment comprising a tag protein.

[0090] More preferably, the tag protein is selected from the group consisting of His, glutathione S-transferases (GSTs), C-myc, chitin binding domain, maltose binding protein (MBP), SUMO heteroaffinity 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, histidine affinity tag (HAT), polyhistidine.

[0091] In one embodiment, the tag protein is located at the N-terminus of the single chain polypeptide.

[0092] In one embodiment, the tag protein is glutathione S-transferases (GSTs).

[0093] Preferably, the first polypeptide segment further comprises a first protease cleavage site.

[0094] Preferably, neither the first protease cleavage site nor the second protease cleavage site can be cleaved by human proteases or proteases produced by the host cell expressing the single chain polypeptide.

[0095] More preferably, the first protease cleavage site and the second protease cleavage site are the same or different.

[0096] More preferably, the specific protease is selected from the group consisting of one or both of non-human enteropeptidase, tobacco etch virus protease, protease derived from Bacillus subtilis, protease derived from Bacillus amyloliquefaciens, protease derived from rhinovirus, papain, insect homolog of papain, or crustacean homolog of papain.

[0097] In one embodiment, the protease that specifically recognizes the first protease cleavage site and the protease that specifically recognizes the second protease cleavage site are both proteases derived from rhinovirus.

[0098] Preferably, the second enzyme cleavage site is embedded in, partially replaces, or completely replaces the activation loop between the first functional peptide segment and the second functional peptide segment. The embedding refers to the insertion between two amino acids of the activation loop; the partial replacement refers to the replacement of part of the amino acid sequence of the activation loop by the second enzyme cleavage site; and the complete replacement refers to the complete replacement of the amino acid sequence of the native activation loop by the second enzyme cleavage site.

[0099] More preferably, the first and second protease cleavage sites are selected from the group consisting of DDDDK, EXXYXQS / G, HY, YH, or a combination of one or two of LEVLFQGP.

[0100] In one embodiment, the first and second protease cleavage sites are both LEVLFQGP.

[0101] Preferably, the first polypeptide fragment further comprises a linker peptide.

[0102] Preferably, the linker peptide makes the first protease cleavage site more easily recognized or bound by its protease.

[0103] More preferably, the linker peptide does not affect the function of the single-chain polypeptide.

[0104] More preferably, the linker peptide remaining at the N-terminus of the second polypeptide fragment after the first protease cleavage site is cleaved does not affect the function of the second polypeptide fragment.

[0105] More preferably, the linker peptide is no more than 5 amino acid residues, and more preferably, the linker peptide is selected from the group consisting of Glycine-Serine (GS) linker peptides, GGS, GGGS, GGGGS, GSGS, GGSGS, GSGGS, GGSGS, GGGSS, etc.

[0106] In one embodiment, the combination of the structural region comprising the first protease cleavage site and the linker peptide is LEVLFQGPLGS.

[0107] In one embodiment, the polypeptide comprises, in order from the N-terminus, glutathione S-transferase, LEVLFQGPLGS, the light chain of BoNT / A, LEVLFQGP, and the heavy chain of BoNT / A. More preferably, the sequence of the polypeptide is shown in SEQ ID NO: 1.

[0108] After the polypeptide is cleaved by the first protease to remove the tag protein, it becomes a polypeptide derivative without the tag protein, and if the second protease cleavage site is recognized and cleaved by a specific protease, a toxin polypeptide with a dimeric structure formed by the light chain and the heavy chain connected by a disulfide bond is formed, which has similar neurotoxicity to the natural toxin molecule. The molecule of the light chain and the heavy chain of the single-chain polypeptide before being cleaved is the precursor of the toxin polypeptide, and the precursor molecule, the polypeptide or the polypeptide derivative, has no toxicity or only slight toxicity compared to the natural molecule.

[0109] The term "polypeptide" as used herein refers to a naturally occurring or genetically engineered polypeptide, preferably from a naturally occurring or genetically engineered toxin polypeptide precursor.

[0110] The toxicity of the polypeptide, i.e. the activity of the polypeptide.

[0111] The term "activation" refers to the contact of the protease, also called activating enzyme, with the low-toxicity toxin polypeptide precursor, the peptide bond between the functional amino acid structure regions is cleaved, the functional amino acid structure regions are connected by disulfide bond, and the toxic polypeptide with spatial conformation and physiological activity is folded.

[0112] The term "functional amino acid structure region" can be understood as a peptide chain known to those skilled in the art, such as light chain, heavy chain or subunit, etc.

[0113] The term "tag protein" is a term known to those skilled in the art, which is a heterologous affinity substance.

[0114] The term "full sealing" as used herein has two aspects of meaning. On the one hand, the sealing function of the equipment pipeline itself, such as rubber gasket, bolt, stainless steel interface, etc. makes the cell lysate processing equipment not leak during operation. On the other hand, the design principle of the cell lysate processing equipment makes it unnecessary to disconnect the equipment for manual liquid change during operation.

[0115] The term "isolator" refers to a conventional device in the art.

[0116] The system of the present application is designed according to the low toxicity of polypeptide, and can greatly reduce the pollution of micro liquid particles into the surrounding environment compared with the traditional centrifugal process. The first filtration treatment of the system can be selected from a plurality of different filter pore diameters, and when the first filtration treatment is completed, the cell lysate enters the circulating liquid storage device and is subjected to the second filtration treatment by pressure. For the problems of filter membrane blockage, liquid replacement and flushing commonly encountered in cell lysate treatment, the above-mentioned device can improve the yield of target protein on one hand, and the waste liquid obtained by the system almost does not contain low-toxicity toxin polypeptide precursor, which has very small toxicity to the operating environment, and can be directly discharged after simple disinfection. Preferably, in the cell lysate device of the present application, the following methods are used in a fully closed manner: on the one hand, when visible deposition occurs in the crude liquid filtration device and the passing rate decreases obviously, the liquid in the liquid inlet pipeline is switched from the cell lysate to the supplement liquid, and the supplement liquid makes the deposition on the crude liquid filtration device separate and enter the filter residue discharge pipeline connected with the crude liquid filtration device; on the other hand, when the turbidity of the liquid reaches a certain degree, the valve on the pipeline branch at the end of the liquid circulating storage device leading to the waste liquid discharge pipeline is opened, and the liquid is discharged as waste liquid. This device not only prevents the leakage of toxic proteins from the beginning of protein crude purification, but also enables the processing of E. coli lysate to be produced only in a fully closed pipeline, without the need for the use of an isolator. This filtration and purification device combined with the low toxicity of polypeptide can avoid the use of an isolator for the safe treatment and preliminary purification of a large amount of toxic protein solution, thereby greatly reducing the production cost under the condition of ensuring safety. At the same time, the multiple chromatography steps further improve the purity of the polypeptide to more than 90%. The above improvements of the system of the present application make it possible to produce large-scale commercial production of toxic polypeptides by recombinant proteins. BRIEF DESCRIPTION OF DRAWINGS

[0117] Figure 1 : Structure diagram of the polypeptide preparation system of the present application.

[0118] Figure 2 : SDS-PAGE results of GSTs-BoNT / A obtained by the system of the present application (lane 1) and 150KD BoNT / A after removing the GSTs tag (lane 2).

[0119] Figure 3 : SDS-PAGE results of the product after removing the GSTs tag, which is further purified by ion exchange column to obtain high-purity BoNT / A protein. DETAILED DESCRIPTION

[0120] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions 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, but all such modifications and substitutions fall within the protection scope of the present invention.

[0121] Example 1: System for preparing peptides

[0122] like Figure 1 As shown, the system for preparing peptides comprises, in sequence:

[0123] (1) Fermentation module: It includes a constant temperature shaker 110, a fermentation device 120, a sealed storage device 130, a pyrolysis device 140, and a homogenizing device 150 in sequence. The sealed storage device 130 is also equipped with a buffer supply device I160, and the pyrolysis device 140 is also equipped with a waste inlet / outlet device 170. The above devices are connected by pipelines, and pumps 400 are installed on the pipelines. The pumps 400 are respectively installed between the constant temperature shaker 110 and the fermentation device 120, between the fermentation device 120 and the sealed storage device 130, between the sealed storage device 130 and the pyrolysis device 140, between the pyrolysis device 140 and the homogenizing device 150, and between the sealed storage device 130 and the buffer supply device I160.

[0124] (2) Enrichment module: the enrichment module here refers to a cell lysate processing device, which comprises a crude liquid filtration device 210, a feed liquid circulation storage device 221, a feed liquid circulation filtration device 222, a waste residue discharge pipeline 231, a waste liquid discharge pipeline 232, a waste collection device 233, a finished product liquid collection device 241, a real-time turbidity monitoring and valve device 242, and a buffer supply device 180; all devices are connected by pipelines, wherein the upstream end of the crude liquid filtration device 210 is connected to the upstream fermentation module and the buffer supply device 180, the crude liquid filtration device 210 is connected to the waste residue discharge pipeline 231, the end of the crude liquid filtration device 210 is connected to the feed liquid filtration device formed by the pipeline connection between the feed liquid circulation storage device 221 and the feed liquid circulation filtration device 222; the end of the feed liquid circulation storage device 221 is also connected to the waste liquid discharge pipeline 232, and the feed liquid circulation filtration device 222 is also connected to the finished product liquid collection flow path, which is composed of the real-time turbidity monitoring and valve device 242 and the finished product liquid collection device 241; a pump is arranged on the pipeline at the upstream end of the crude liquid filtration device 210 and on the pipeline between the end of the feed liquid circulation storage device 221 and the upstream end of the feed liquid circulation filtration device 222; a pipeline valve is arranged on the pipeline leading to the waste liquid discharge pipeline 232 at the end of the feed liquid circulation storage device 221; all devices are connected by pipelines; the finished product liquid collection device 241 is connected to the downstream purification module; the pore size of the crude liquid filtration device 210 is 0.1-0.65 μm, and the pore size of the filter material in the feed liquid circulation filtration device 222 is 0.2 μm or less, and the material can be any hydrophilic or hydrophobic filter material.

[0125] (3) Purification module: sequentially comprising a chromatography device I 311, a chromatography device II 312, and a chromatography device III 313; the upstream end of the chromatography device I 311 is provided with a protease liquid supply device 320; the downstream end of the chromatography device III 313 is provided with a finished product collection device 330; the upstream end of each chromatography device is provided with a cleaning liquid supply device 341-344 and a chromatography buffer supply device 351-352; the downstream end of each chromatography device is provided with a waste liquid collection device 361-363; an isolator 370 is arranged outside each chromatography device; the protease liquid supply device 320 can also comprise two or more, and the protease liquid comprises different proteases;

[0126] The (1), (2), and (3) are connected by pipelines.

[0127] Example 2: Expression of GSTs-BoNT / A

[0128] A host cell expressing a polypeptide of interest is prepared using genetic engineering techniques (Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989), including: 1. designing and constructing a nucleic acid molecule encoding a modified neurotoxin polypeptide; 2. constructing a plasmid containing the nucleic acid molecule of step 1; 3. transfecting the plasmid constructed in step 2 into a host cell; 4. inoculating, culturing, fermenting, lysing and homogenizing the E. coli expressing the polypeptide using the fermentation module in the system of the present application.

[0129] wherein the expressed polypeptide has an amino acid sequence as shown in SEQ ID NO: 1.

[0130] Example 3: Enrichment of GSTs-BoNT / A

[0131] The product cell lysate crude liquid of Example 2 is enriched using the system of Example 1: it is pumped into the fully-closed enrichment module, i.e. the fully-closed cell lysate separation and purification instrument. In the fully-closed cell lysate separation and purification instrument, the lysate and residue are first separated by the first layer of crude liquid filtration device, the residue enters the waste recovery device, and the filtered lysate is further used as the feed liquid to enter the feed liquid circulation filtration device. During the circulation filtration process, the circulation liquid that meets the product liquid collection conditions enters the product liquid collection device, and the circulation liquid that does not meet the product liquid collection conditions continues to be circulated and filtered or enters the waste liquid recovery device.

[0132] In view of the common problems of filter membrane blockage, liquid change and flushing during cell lysate processing, in the cell lysate equipment described in the present application, on the one hand, when visible deposition occurs in the crude liquid filtration device and the passage rate decreases significantly, the liquid in the liquid inlet pipeline is switched from the cell lysate to the buffer II, which causes the deposition on the crude liquid filtration device to detach and enter the filter residue discharge pipeline connected to the crude liquid filtration device; on the other hand, when the turbidity of the feed liquid reaches a certain degree, the valve on the pipeline branch at the end of the feed liquid circulation storage device leading to the waste liquid discharge pipeline is opened, and the feed liquid is discharged as waste liquid. No manual liquid change and flushing operations are required throughout the process.

[0133] The product liquid collection conditions are that the circulation is performed at least twice.

[0134] The product liquid obtained by the fully-closed cell lysate separation and purification instrument contains more than 90% of the polypeptide, the waste liquid obtained almost does not contain single-chain polypeptide, and has very little toxicity to the operating environment, which can be directly discharged after simple disinfection.

[0135] Example 4: Cleavage of GSTs tag protein and purification of BoNT / A and BoNT / A

[0136] The product of Example 2, after enrichment module, enters the purification module, and the product of Example 2 is contacted with GSTs affinity ligand in the first chromatographic device, affinity chromatographic device, and the material not bound to the ligand is eluted by using conventional method, and the GSTs is dissociated from the ligand by using conventional method, and the preliminary purified GSTs-BoNT / A is obtained.

[0137] The conventional method is as follows: the chromatographic column is washed with 20 times column volume of phosphate buffer, and 10 times column volume of freshly prepared 10 mM glutathione elution buffer (0.154 g reduced glutathione is dissolved in 50 ml 50 mM Tris-HCl (pH 8.0) to elute GSTs-BoNT / A, and the elution of the fusion protein is monitored by using absorbance reading at 280 nm.

[0138] The GSTs tag protein is cleaved under the action of Rinovirus 3C Proteas after elution.

[0139] As shown in Figure 2 : compared with the first lane without tag protein removal, the GSTs tag protein in the first lane is cleaved under the action of Rinovirus 3C Protease, and the BoNT / A molecule without GSTs is obtained.

[0140] The preliminary purified product is further purified in the second chromatographic device, gel filtration chromatographic device, or the third chromatographic device, ion chromatographic device, and the purification method is a method known to those skilled in the art, and the results are shown in Figure 3 : the band corresponding to the polypeptide is obviously thickened, and the BoNT / A molecule with a purity of 90% can be obtained.

[0141] Example 5: Toxicity experiment of GSTs-BoNT / A and BoNT / A

[0142] The LD of the GSTs-BoNT / A obtained in Example 4 injected into the abdominal cavity of mice 50 is between 45 ng and 450 ng; considering the purity of the botulinum toxin protein injected, the converted LD 50 is between 22.5 ng and 225 ng, and the median value is 123.75 ng. The LD of the BoNT / A obtained in Example 4 injected into the abdominal cavity of mice 50 is between 0.02 ng and 0.05 ng. Considering the purity of the botulinum toxin protein injected, the converted LD 50 is between 0.006 ng and 0.015 ng, and the median value is 0.0105 ng (see Table 1).

[0143] Table 1. Toxicity comparison of GSTs-BoNT / A and BoNT / A molecules in mouse bio toxicity experiment

[0144]

[0145] GSTs-BoNT / A has the activity of botulinum toxin, and after intraperitoneal injection in mice, its median lethal dose (LD 50 ) is about 11786 times higher than the protein LD 50 of BoNT / A, indicating that the activity of GSTs-BoNT / A recombinant protein toxin precursor molecule is about 11786 times weaker than the final product BoNT / A. This experiment proves that the GSTs-BoNT / A recombinant protein toxin precursor molecule has the activity of botulinum toxin, but its toxicity is much lower than that of activated BoNT / A. Therefore, the cell lysate separation device containing the polypeptide of the toxin precursor can be treated with full sealing, that is, the sealing function of the equipment pipeline itself, such as rubber gasket, bolt, stainless steel joint, and during operation, manual flushing, liquid change and other operations are not required. The full-sealed cell lysate treatment equipment can completely process the cell lysate containing the low-toxicity toxin polypeptide precursor without the need for additional sealed work space, such as isolator.

[0146] Through the examples of the present application, it is proved that the system for preparing polypeptides claimed in the present application can be applied to the preparation of toxic polypeptides, which exist in the form of low toxicity in host cells and cell lysates, and are activated to toxin molecules with natural toxicity only after specific protease hydrolysis. In this case, whether it is cell fermentation, lysis or lysate separation, it can be carried out in the closed system of the present application, without isolator in these steps, on the one hand, improving the safety of low-toxicity polypeptide production, and on the other hand, reducing the cost of production. At the same time, the multi-step chromatography purification after protease activation further improves the purity of active polypeptide, which can reach 90%, thus making large-scale industrial production of such polypeptides possible.

[0147] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, which all belong to the protection scope of the present application.

[0148] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combinations. SEQUENCE LISTING <110> Suzhou Yuyan Biotechnology Co., Ltd. <120> A system for preparing polypeptides and uses thereof <130> 1 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1524 <212> PRT <213> Artificial Sequence <400> 1 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu 210 215 220 Phe Gln Gly Pro Leu Gly Ser Met Pro Phe Val Asn Lys Gln Phe Asn 225 230 235 240 Tyr Lys Asp Pro Val Asn Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro 245 250 255 Asn Ala Gly Gln Met Gln Pro Val Lys Ala Phe Lys Ile His Asn Lys 260 265 270 Ile Trp Val Ile Pro Glu Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly 275 280 285 Asp Leu Asn Pro Pro Pro Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr 290 295 300 Asp Ser Thr Tyr Leu Ser Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys 305 310 315 320 Gly Val Thr Lys Leu Phe Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg 325 330 335 Met Leu Leu Thr Ser Ile Val Arg Gly Ile Pro Phe Trp Gly Gly Ser 340 345 350 Thr Ile Asp Thr Glu Leu Lys Val Ile Asp Thr Asn Cys Ile Asn Val 355 360 365 Ile Gln Pro Asp Gly Ser Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile 370 375 380 Ile Gly Pro Ser Ala Asp Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly 385 390 395 400 His Glu Val Leu Asn Leu Thr Arg Asn Gly Tyr Gly Ser Thr Gln Tyr 405 410 415 Ile Arg Phe Ser Pro Asp Phe Thr Phe Gly Phe Glu Glu Ser Leu Glu 420 425 430 Val Asp Thr Asn Pro Leu Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro 435 440 445 Ala Val Thr Leu Ala His Glu Leu Ile His Ala Gly His Arg Leu Tyr 450 455 460 Gly Ile Ala Ile Asn Pro Asn Arg Val Phe Lys Val Asn Thr Asn Ala 465 470 475 480 Tyr Tyr Glu Met Ser Gly Leu Glu Val Ser Phe Glu Glu Leu Arg Thr 485 490 495 Phe Gly Gly His Asp Ala Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu 500 505 510 Phe Arg Leu Tyr Tyr Tyr Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu 515 520 525 Asn Lys Ala Lys Ser Ile Val Gly Thr Thr Ala Ser Leu Gln Tyr Met 530 535 540 Lys Asn Val Phe Lys Glu Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly 545 550 555 560 Lys Phe Ser Val Asp Lys Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu 565 570 575 Thr Glu Ile Tyr Thr Glu Asp Asn Phe Val Lys Phe Phe Lys Val Leu 580 585 590 Asn Arg Lys Thr Tyr Leu Asn Phe Asp Lys Ala Val Phe Lys Ile Asn 595 600 605 Ile Val Pro Lys Val Asn Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg 610 615 620 Asn Thr Asn Leu Ala Ala Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn 625 630 635 640 Asn Met Asn Phe Thr Lys Leu Lys Asn Phe Thr Gly Leu Phe Glu Phe 645 650 655 Tyr Lys Leu Leu Cys Val Arg Gly Ile Ile Thr Ser Leu Glu Val Leu 660 665 670 Phe Gln Gly Pro Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp 675 680 685 Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn 690 695 700 Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu 705 710 715 720 Asn Ile Ser Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe 725 730 735 Asp Asn Glu Pro Glu Asn lie Ser lie Glu Asn Leu Ser Ser Asp lie 740 745 750 Ile Gly Gin Leu Glu Leu Met Pro Asn lie Glu Arg Phe Pro Asn Gly 755 760 765 Lys Lys Tyr Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala 770 775 780 Gln Glu Phe Glu His Gly Lys Ser Arg lie Ala Leu Thr Asn Ser Val 785 790 795 800 Asn Glu Ala Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser 805 810 815 Asp Tyr Val Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu 820 825 830 Gly Trp Val Glu Gin Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu 835 840 845 Val Ser Thr Thr Asp Lys lie Ala Asp lie Thr lie lie lie Pro Tyr 850 855 860 Ile Gly Pro Ala Leu Asn lie Gly Asn Met Leu Tyr Lys Asp Asp Phe 865 870 875 880 Val Gly Ala Leu lie Phe Ser Gly Ala Val lie Leu Leu Glu Phe lie 885 890 895 Pro Glu Ile Ala Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr 900 905 910 Ile Ala Asn Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser 915 920 925 Lys Arg Asn Glu Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn 930 935 940 Trp Leu Ala Lys Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met 945 950 955 960 Lys Glu Ala Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn 965 970 975 Tyr Gln Tyr Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe 980 985 990 Asn Ile Asp Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala 995 1000 1005 Met Ile Asn Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu 1010 1015 1020 Met Asn Ser Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp 1025 1030 1035 1040 Ala Ser Leu Lys Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly 1045 1050 1055 Thr Leu Ile Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr 1060 1065 1070 Leu Ser Thr Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln 1075 1080 1085 Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr 1090 1095 1100 Ser Ile Leu Asn Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser 1105 1110 1115 1120 Arg Tyr Ala Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro 1125 1130 1135 Ile Asp Lys Asn Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile 1140 1145 1150 Glu Val Ile Leu Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn 1155 1160 1165 Phe Ser Thr Ser Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile 1170 1175 1180 Ser Leu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser 1185 1190 1195 1200 Gly Trp Lys Val Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln 1205 1210 1215 Asp Thr Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met 1220 1225 1230 Ile Asn Ile Ser Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr 1235 1240 1245 Asn Asn Arg Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile 1250 1255 1260 Asp Gln Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn 1265 1270 1275 1280 Ile Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp 1285 1290 1295 Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile 1300 1305 1310 Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe 1315 1320 1325 Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu 1330 1335 1340 Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly 1345 1350 1355 1360 Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile 1365 1370 1375 Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys 1380 1385 1390 Tyr Ala Ser Gly Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val 1395 1400 1405 Tyr Ile Asn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn 1410 1415 1420 Ala Ser Gln Ala Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro 1425 1430 1435 1440 Asp Val Gly Asn Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp 1445 1450 1455 Gln Gly Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly 1460 1465 1470 Asn Asp Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys 1475 1480 1485 Leu Val Ala Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg 1490 1495 1500 Thr Leu Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly 1505 1510 1515 1520 Glu Arg Pro Leu

Claims

1. A system for preparing a polypeptide, characterized in that, The system comprises an enrichment module, The enrichment module comprises a cell lysate separation device, which is fully enclosed, and the cell lysate separation device comprises a multiple filtration device and a waste discharge pipeline connected to the multiple filtration device; the multiple filtration device comprises a crude liquid filtration device and a feed liquid filtration flow path, and the front end of the crude liquid filtration device is connected to a lysate inlet pipeline and a buffer inlet pipeline; The system comprises a purification module downstream of the enrichment module, and the purification module comprises a protease liquid supply device; The polypeptide exists in the form of a low-toxicity single-chain polypeptide in the cell or lysate and has toxicity after a downstream protease activation step; The feed liquid filtration flow path comprises a feed liquid circulation filtration device and a feed liquid circulation storage device; The polypeptide is a genetically recombined neurotoxin precursor; The purification module comprises at least one set of chromatography device; The protease liquid supply device is connected to the front end of the first chromatography device.

2. The system of claim 1, wherein, The waste discharge pipeline comprises a waste residue discharge pipeline and a waste liquid discharge pipeline, the waste residue discharge pipeline is arranged downstream of the crude liquid filtration device, and the waste liquid discharge pipeline is arranged downstream of the feed liquid filtration flow path.

3. The system of claim 1, wherein, The lysate inlet pipeline and the buffer inlet pipeline are the same or different.

4. The system of claim 2, wherein, The feed liquid circulation filtration device and the feed liquid circulation storage device form a loop, and the waste liquid discharge pipeline is arranged at the end of the feed liquid circulation storage device.

5. The system of claim 1, wherein, The pore size of the filter material of the crude liquid filtration device is suitable for the separation of solids and liquids in the cell lysate.

6. The system of claim 1, wherein, The pore size of the filter material of the crude liquid filtration device is 0.1-0.65 μm.

7. The system of claim 1, wherein, The pore size of the filter material in the feed liquid circulation filtration device is 0.2 μm or less.

8. The system of claim 1, wherein, The filter material of the multiple filtration device is selected from hydrophilic material or hydrophobic material, the hydrophilic material is selected from cellulose ester, polyether sulfone or derivatives thereof, and the hydrophobic material is selected from polyvinylidene fluoride, polypropylene, polyethylene, polytetrafluoroethylene or derivatives thereof.

9. The system of claim 1, wherein, The system further comprises a fermentation module upstream of the enrichment module.

10. The system of claim 9, wherein, The fermentation module comprises a host cell culture device, a fermentation device, and a lysis device.

11. The system of claim 10, wherein, The lysis device is arranged downstream of the culture device.

12. The system of claim 10, wherein, The fermentation module further comprises a sealed storage device for host cell fermentation liquid, and the sealed storage device is arranged between the culture device and the lysis device.

13. The system of claim 10, wherein, The fermentation module further comprises a homogenization device arranged downstream of the lysis device.

14. The system of claim 1, wherein, The purification module comprises three sets of chromatography devices.

15. The system of claim 1, wherein, The chromatography devices are located in isolators.

16. The system of claim 1, wherein, The chromatography devices are affinity chromatography devices, gel filtration chromatography devices, and ion chromatography devices.

17. Use of the system according to any one of claims 1 to 16 for the preparation of a polypeptide, characterized in that, The polypeptide exists in the form of a low-toxicity single-chain polypeptide in the cell or lysate and has toxicity after a downstream protease activation step.

18. The use of claim 17, wherein, The polypeptide comprises at least two functional amino acid domains, wherein the first functional amino acid domain comprises a metal ion-dependent protease activity domain, the second functional amino acid domain comprises a receptor binding domain that binds to a target cell surface receptor and / or a translocation domain that mediates the translocation of the polypeptide across a vesicle membrane, and the two functional amino acid domains are connected by a domain comprising a protease cleavage site.

19. The use according to claim 18, wherein the compound is ###00010### 18 The protease cleavage site is a 3C enzyme cleavage site.

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