WO2017143839A1

WO2017143839A1 - Method for synthesis of pharmaceutical recombinant protein based on intein

Info

Publication number: WO2017143839A1
Application number: PCT/CN2016/110291
Authority: WO
Inventors: 陈俊生; 朱建伟; 韩雷; 丁凯; 谢跃庆; 江华; 路慧丽; 张宝红; 张蕾
Original assignee: 上海交通大学; 美国杰科实验室有限公司; 杰科（天津）生物医药有限公司
Priority date: 2016-02-23
Filing date: 2016-12-16
Publication date: 2017-08-31
Also published as: CN105925596A

Abstract

Disclosed is a method for the synthesis of a pharmaceutical recombinant protein based on an intein, comprising the following steps: construction of vector DNA fragments expressing a polypeptide having a targeting function and a polypeptide having a toxic function in an immunotoxin, the vector DNA fragments of the two polypeptides respectively being linked to the N-terminus or C-terminus of the intein to form a fusion expression vector; taking the fusion expression vector described in step 1, and expressing same to obtain a fusion polypeptide A that fuses the N-terminus of the intein and a fusion polypeptide B that fuses the C-terminus of the intein; taking the fusion polypeptide A and the fusion polypeptide B, mixing them, and inducing the mixture under trans-splicing conditions corresponding to the intein to obtain the immunotoxin. The method only requires the preparation of different targeting part polypeptides and toxic part polypeptides, wherein same can be combined and then produce an immunotoxin that can target different targets and have different toxic mechanisms, with significant diversity and flexibility.

Description

Method for synthesizing medicinal recombinant protein based on intein

Technical field

The present invention relates to a method for synthesizing a pharmaceutical recombinant protein in the field of biotechnology, and in particular to a method for synthesizing a pharmaceutical recombinant protein based on an intein.

Background technique

An immunotoxin is a fusion protein comprising a targeting polypeptide moiety, a toxic polypeptide moiety, and/or a linker polypeptide moiety. Since the monoclonal antibody was first prepared by Kohlor's hybridoma technology in 1957, it has shown broad application prospects in medical research and clinical diagnosis and treatment of diseases. For a long time, people have been working on the use of monoclonal antibodies to treat a variety of diseases, such as tumors, autoimmune diseases, etc., but the effect of using monoclonal antibodies alone is sometimes not ideal. In order to achieve a more effective therapeutic effect, some cytotoxic proteins are combined with monoclonal antibodies to form an "immunotoxin" with selective killing of cells combined with it, and equipped with an ammunition depot for targeted therapy of diseases. New ammunition.

Immunotoxins (ITs) are protein molecules produced by combining a protein with a targeting function and a toxin protein. The part with guiding function is mainly responsible for guiding the specific binding of the immunotoxin protein molecule to the target cell, while the toxin protein part mainly plays a role in killing the cell.

From the perspective of the evolution of immunotoxins, the preparation and production of immunotoxins mainly include chemical coupling and recombinant expression. The preparation of immunotoxins by chemical coupling first requires the preparation of antibodies and toxins separately, followed by chemical coupling to form an immunotoxin. The chemical coupling method has low coupling efficiency, high production cost, and poor product uniformity due to a large number of sites on the protein where coupling reaction may occur, and the coupled chemical bond tends to degrade during circulation in the body, making the naked toxin Leakage leads to non-specific toxicity, and there is a greater risk of side effects, while naked antibodies produced by degradation may block the antigen, resulting in poor therapeutic effect. With the development of genetic engineering, the preparation and production of immunotoxins has entered a new era. Genes encoding a functional polypeptide are fused to a gene encoding a toxin polypeptide and expressed in an appropriate expression system using genetic recombination techniques. The immunotoxin produced by this technical scheme can be called genetic engineering immunotoxin, which has greatly improved product homogeneity and stability compared with the immunotoxin produced by chemical coupling method, and makes mass production immunity. Toxins are possible. However, the genetic engineering method for the production of immunotoxins has its limitations: the fusion gene is restricted to a single host for expression, and constitutes The contradiction between the targeting part and the toxic part of the immunotoxin often requires different host expression environments, which often leads to the inability to obtain good yield, yield, purity and consequent cost increase for the immunotoxin using a single host. For example, E. coli expression systems are currently used to express single-chain antibody immunotoxins. Because the targeting part of immunotoxins does not fold well in E. coli expression systems, inclusion bodies are often formed, and the refolding of inclusion bodies is a very complicated process. In general, the renaturation efficiency of proteins is about 20%; while toxin proteins are lethal to eukaryotic cells. If eukaryotic expression systems are used, they may be toxic to host cells, but some researchers use eukaryotic expression systems. The expression of immunotoxins has done a great deal of work. For example, patent CN1863921 discloses a method for expressing immunotoxins in Pichia pastoris and EF-2 mutant Pichia pastoris, although in the manner of secretory expression in Pichia pastoris and EF-2 The mutant (toxin-immunized) Pichia pastoris expression system successfully expressed the immunotoxin, and the lower yield obtained by the longer fermentation cycle is not competitive with the prokaryotic expression system, and the sugar on the toxin protein The basement site may be glycosylated by the host, which may introduce product heterogeneity; the literature discloses a A method for expressing immunotoxins in F-2 mutant CHO cells, which also suffers from low expression levels, long fermentation cycles, high cost, and potential glycosylation (Protein expression and purification, 2000, 19(2) :304-311).

Intein refers to a sequence present in a precursor protein. During the conversion of a precursor protein into a mature protein, the exopeptide at both ends of the intein is linked by a peptide bond by self-splicing, and the precursor is itself The protein is released. The intein can be divided into N-terminus and C-terminus both structurally and functionally. When the N-terminus or C-terminus are present alone, the splicing reaction cannot occur, and only when the N-terminus and the C-terminus are contacted under conditions suitable for splicing, splicing can occur. reaction. The cleavage intein may be artificially cleavage of the continuous intein from the appropriate position into two polypeptide fragments, or may be naturally occurring. Naturally cleavable inteins are a class of two polypeptide fragments encoded by two separately transcribed and expressed genes that self-assemble upon contact and catalyze the attachment of the exopeptide in a trans-splicing manner. Inteins are widely used in biotechnology applications, including intein-mediated protein ligation (IPL), protein cyclization, protein labeling, toxic protein expression, introduction of unnatural amino acids, studies using intein splicing activity In vivo protein interactions, etc. The use of inteins and their variants to mediate protein purification and their use in large-scale protein production is also receiving increasing attention. Therefore, there is a need in the art to provide a method that is versatile, easy to operate, and inexpensive to obtain a fusion immunotoxin.

Summary of the invention

The object of the present invention is to overcome the deficiencies of the prior art and to provide a method for synthesizing a pharmaceutical recombinant protein based on inteins. The invention overcomes the existing product unevenness in the preparation of immunotoxin technology by chemical coupling or fusion expression method 1. Insufficient operation steps, toxicity of the target protein to the expression host, etc., providing a separately prepared polypeptide having a targeting function and a cytotoxic polypeptide and linked together by the trans-splicing function of the intein, thereby A method of obtaining an immunotoxin.

The present invention is achieved by the following technical scheme, and relates to a method for synthesizing a pharmaceutical recombinant protein based on intein, the method comprising the following steps:

Step one: constructing a vector DNA fragment expressing a polypeptide having a guiding function in an immunotoxin and a polypeptide having toxic function, and connecting the vector DNA fragment of the two polypeptides to the N-terminus or the C-terminus of the intein to form a fusion expression. a vector; wherein the polypeptide expressed by the fusion expression vector containing the N-terminus of the intein and the fusion expression vector containing the C-terminus of the intein is required to be paired so that the trans-splicing reaction can occur;

Step 2, the fusion expression vector described in the first step is separately expressed in a suitable host cell to obtain a fusion polypeptide A fused to the N-terminus of the intein and a fusion polypeptide B fused to the C-terminus of the intein;

In the third step, the fusion polypeptide A and the fusion polypeptide B are taken, mixed, and the polypeptide which induces the guiding function of the immunotoxin and the polypeptide part having the toxic function are trans-spliced under the trans-splicing condition corresponding to the intein to obtain an immunotoxin.

Preferably, in the first step, the intein is an artificially-cleaved intein, or a naturally-cleaved intein, or an intein mutant having a splicing function.

Preferably, in step 1, the intein is Ssp DnaB, Ssp DnaE or Npu DnaE.

Preferably, in the first step, the N-terminus of the N-terminus of the intein or the N-terminus of the C-terminus is further fused with a functional polypeptide.

Preferably, the functional polypeptide is an MBP or ChBD tagged protein.

Preferably, in the first step, the fusion expression vector is configured to direct the functional polypeptide-intein N-terminus, or the intein C-terminal toxic functional polypeptide, or the toxic functional polypeptide-intein N-terminus, or the inclusion a peptide C-terminally functional polypeptide, or a targeting functional polypeptide-intein N-terminal functional polypeptide, or a functional polypeptide-intein C-terminal toxic functional polypeptide, or a toxic functional polypeptide-intein N-terminal functional polypeptide, Functional polypeptide-intein C-terminal-directed functional polypeptide.

Preferably, in step 1, the polypeptide having a targeting function is an antibody, a single-chain antibody scFv, a disulfide-stabilized antibody dsFv, a disulfide-stabilized single-chain antibody dsscFv, a Fab antibody, a cytokine, or a growth factor.

Preferably, in the first step, the toxic functional polypeptide is a RIP type toxin; specifically, a fusion protein of a ricin toxin, an ADP ribosylation immunotoxin, a diphtheria toxin, or a Pseudomonas aeruginosa exotoxin portion, or a toxin organism Active mutant.

Preferably, in step two, the host cell is a prokaryotic cell or a eukaryotic cell.

Preferably, the host cell is Escherichia coli, Bacillus subtilis, high expansion yeast cell, insect cell, plant cell, mammalian cell, yeast, Chinese hamster ovary cell, or human kidney blast cell.

Preferably, in the third step, the step of separating and purifying to obtain an immunotoxin is further included.

Preferably, the method of separation and purification is an affinity chromatography method, an ion exchange chromatography method, or a hydrophobic interaction chromatography method.

Preferably, in step three, the trans-splicing condition is a condition capable of triggering the completion of self-splicing of the specified intein.

Preferably, when the intein is Npu DnaE, the trans-splicing conditions are: 0-55 ° C, pH 3-11, contact time 1 s - 60 h, final concentration of 0.05 mM - 100 mM nucleophile.

Preferably, the nucleophile is DTT, DTE, CYSTEINE, TCEP, or MESNA nucleophilic reagent.

It is further known to those skilled in the art that in the preparation method of the present invention, in step 1, the intein is an intein having a self-splicing function, which may be wild type or may comprise a variation relative to the wild type, preferably An intein having a trans-splicing function, such as an artificially-cleaved intein, a naturally-cleaved intein, preferably a naturally-cleaved intein such as Ssp DnaB, Ssp DnaE or Npu DnaE, preferably Npu DnaE; In an embodiment of the peptide-containing splicing function, the linker between the intein and the protein of interest may comprise a natural exopeptide sequence. The term "exopeptide" as used herein refers to a sequence found in nature that is adjacent to an intein or intein domain, for example, corresponding to wild-type Npu DnaE, and the C-terminal exopeptide may be CFNAS, CFNK, CFN, CF. , C. In the first step, the toxic functional polypeptide may include a RIP type toxin such as ricin, an ADP ribosylation immunotoxin, such as a diphtheria toxin, a fusion protein of a Pseudomonas aeruginosa exotoxin moiety, and the like. The toxin moiety can be a truncated portion and/or can comprise a variation relative to a wild-type toxin. In the first step, the expression vector is a variety of expression vectors or modified variants thereof known to those skilled in the art, and a person skilled in the art can obtain a DNA molecule encoding a polypeptide of interest according to conventional means, and is well known in the art. a method, which is linked to an expression control sequence in the expression vector; in the first step, the polypeptide having a targeting function in the immunotoxin may be an antibody, a single-chain antibody scFv, a disulfide-stabilized antibody dsFv, two A sulfur-stable single-chain antibody dsscFv, a Fab antibody, a cytokine, a growth factor, and the like, and a polypeptide having immunoaffinity adsorption ability well known to those skilled in the art, and those skilled in the art can select which type of immunotoxin to use according to the target cell. Peptide.

In the second step, the host cell may include prokaryotic cells and eukaryotic cells, such as commonly used prokaryotic host cells such as Escherichia coli, Bacillus subtilis, etc., commonly used eukaryotic host cells, high expansion yeast cells, insect cells, plant cells, breastfeeding Animal cells, etc. Host cells described in the present invention include, but are not limited to, Escherichia coli, yeast, and Chinese warehousing Mouse ovary cells, human kidney embryo cells, and the like. It is well known to those skilled in the art that there are many methods for transforming/transfecting a host cell with an expression vector, and the transformation method and transformation procedure used depend on the host to be transformed. For example, protoplast fusion, electroporation, liposome-mediated transfection, cationic-mediated transfection, etc.; the host cells obtained by transformation/transfection are cultured under conditions suitable for expression of the polypeptide of interest, and then according to the nature and localization of the polypeptide of interest. Various protein purification steps well known to those skilled in the art, such as affinity chromatography (including but not limited to Protein A, Protein G, Protein L, IMAC, etc.), hydrophobic interaction chromatography, ion exchange chromatography, dialysis, etc., may be selected. Separation and purification means.

In the third step, the separation and purification can be carried out by a conventional protein purification step. In step three, mixing refers to contacting a substance under conditions that are physically associated with another substance.

Compared with the prior art, the present invention has the following beneficial effects: there are many deficiencies in the traditional technical methods for producing immunotoxins, such as the need to add chemical reagents in the chemical coupling method, due to more modification sites in the targeting polypeptide portion. The resulting product is not uniform, and the chemically coupled bond is easily broken, resulting in toxic leakage. In the strategy of directly expressing the immunotoxin fusion protein, if the prokaryotic expression system is used, the target protein often exists in the form of inclusion bodies, renaturation. The inefficiency and cumbersome steps are complicated. If eukaryotic expression systems are used, the expression of immunotoxins may be limited by their natural toxicity to host cells. The method of the present invention overcomes the deficiencies in the traditional strategies of immunotoxin production and achieves unexpected technical effects:

1. The method of the present invention only needs to prepare different targeting partial polypeptides and toxic partial polypeptides, and can be combined to produce immunotoxins which can target different targets and possess different toxicity mechanisms, and have remarkable diversity and flexibility. ;

2. In the method of the present invention, the targeting moiety polypeptide and the toxic moiety polypeptide can be expressed separately in a suitable host cell, such as requiring a special folding environment, particularly advanced post-translational modification, which can be expressed in mammalian cells. Polypeptides that do not have much modification requirements can be expressed in E. coli, and expression of the polypeptide of interest in a suitable expression system can achieve higher yield, yield, and purity;

3. In the method of the present invention, the linkage between the targeting moiety polypeptide and the toxin partial polypeptide is site-specific, does not produce by-products, and the obtained product product has high homogeneity;

4. In the method of the present invention, the self-splicing of the targeting partial polypeptide and the toxin partial polypeptide via the intein is linked by peptide bonds, and has good stability compared to a chemical coupling method;

5. In the method of the invention, the self-splicing reaction condition is mild, the reaction is efficient, and it is easy to integrate and amplify with other processes; the reaction process does not need to add toxic and harmful substances.

DRAWINGS

Other features, objects, and advantages of the present invention will become apparent from the Detailed Description of Description

Fig. 1 is a view showing the results of the fusion protein Nc-PE3KDEL purified by a Ni-Sepharose column by SDS-PAGE in Example 1 of the method for producing an immunotoxin using an intein.

2 is a schematic diagram showing the results of the fusion protein dsscFv CD22-Nn-Fc purified by a Ni-Sepharose column by SDS-PAGE in Example 1 of the method for preparing an immunotoxin using an intein.

Figure 3 is a schematic diagram showing the results of trans-splicing of dsscFv CD22-Nn-Fc and Nc-PE38KDEL to dsscFv CD22-PE38KDEL by Western Blot in Example 1 of the method for preparing an immunotoxin using intein.

Figure 4 is a diagram showing the results of the fusion protein Fab-Nn purified by Protein L chromatography column by SDS-PAGE in Example 2 of the method for preparing an immunotoxin using inteins of the present invention.

Figure 5 is a schematic diagram showing the results of Fb-Nn and Nc-PE38KDEL trans-splicing to generate Fab-PE38KDEL by SDS-PAGE in Example 2 of the method for preparing an immunotoxin using inteins of the present invention.

6 is a schematic diagram showing the results of the method for preparing an immunotoxin by using an intein according to the third embodiment of the method for detecting Herceptin-PE38KDEL by trans-splicing of Herceptin-Nn and Nc-PE38KDEL by Western Blot.

Figure 7 is a structural diagram of an expression plasmid involved in the examples.

detailed description

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Test methods not specified in the following examples are usually carried out according to the conditions described in conventional conditions, such as molecular clones such as Sambrooke: Laboratory Manual, Third Edition (Science Press, 2002), or according to various manufacturers. Recommended conditions.

Example 1. Preparation of immunotoxin dsscFv CD22-PE38KDEL by reverse splicing function of Npu DnaE

1. Construction of a toxic polypeptide expression vector pET-28a(+)-Nc-PE38KDEL fused to the C-terminus of the broken intein Npu DnaE

The gene encoding PE38KDEL toxin and the C-terminal gene of Npu DnaE were cloned by primers in the table, and amplified by PrimalStar Max of TaKaRa. The PCR conditions were 94 ° C for 10 s, 55 ° C for 10 s, 72 ° C for 10 s, 30 cycles. After the fragment was subjected to agarose gel electrophoresis, the C-terminal gene encoding Npu DnaE and the gene encoding PE38KDEL were synthesized by the overlapping PCR at the N-terminus of the fusion polypeptide according to the C-terminus of Npu DnaE. The PCR conditions were 94 ° C for 10 s and 55 ° C for 10 s. This gene fragment was treated with NdeI and NotI at 10 °C for 30 s at 72 ° C, and ligated with pET-28a (+) also treated with NdeI and NotI. The plasmid structure is shown in Fig. 7. The ligation product was transformed into E. coli DH5α competent cells, and the transformed cells were plated in a solution containing 50 μg/mL kanamycin. The lipid plates were incubated overnight. The monoclonal clones on the plate were picked and shaken overnight in 5 mL of LB medium containing 50 μg/mL kanamycin, and the plasmid was extracted and sequenced. The sequencing results showed that the constructed Nc-PE38KDEL sequence was correct.

Table 1

2. Expression and purification of fusion protein Nc-PE38KDEL

The correctly sequenced plasmid was transformed into the competent state of E. coli expression strain BL21, and the single bacteria were cultured overnight at 37 ° C. The single colony was cultured in 5 ml LB medium containing 50 μg/mL kanamycin at 37 ° C, 180 rpm, overnight culture. .

5 ml of the culture solution was added to 500 ml of fresh LB medium containing 50 μg/mL kanamycin, and cultured at 37 ° C until the OD600 was 0.6-0.8. IPTG was added to a final concentration of 0.2 mM, and induced at 30 ° C for 16 h, centrifuged at 4000 rpm. The cells were collected at 10 min.

The collected cells were resuspended in binding buffer (20 mM PBS, 500 mM NaCl, 20 mM imidazole, pH 7.5, 20 ml of binding buffer per 1 g of the cells), and the cells were disrupted by a high-pressure homogenizer, and centrifuged at 12,000 rpm for 30 min at 4 ° C. The supernatant was collected, and the supernatant was filtered with 0.45 μm to prepare for purification with Ni2+NTA.

The Ni ²⁺ gravity column was packed, the volume of the bed was 1 ml, washed with 5 column volumes of water, and then equilibrated with 10 column volume of binding buffer (20 mM PBS, 500 mM NaCl, 20 mM imidazole, pH 7.5), and the collected supernatant was applied. Column, flow rate 1 ml/min, after completion of the upper column, rinse with 5 column volumes of Binding Buffer, followed by elution buffer containing 60 mM imidazole (10 mM PBS, 500 mM NaCl, 60 mM imidazole, pH 7.5) in 10 column volumes The non-specific binding protein was washed, and then eluted with 10 column volumes of 150 mM imidazole in elution buffer (20 mM PBS, 500 mM NaCl, 150 mM imidazole, pH 7.5), collected in a tube, 1 ml per tube, and eluted by SDS-PAGE. In the case, the target protein was combined to obtain the fusion protein Nc-PE38KDEL. Fig. 1 is a view showing the results of the fusion protein Nc-PE3KDEL purified by a Ni-Sepharose column by SDS-PAGE in Example 1 of the method for producing an immunotoxin using an intein.

3. Construction of CD22 antibody expression vector pET-22b(+)-dsscFv CD22-Nn-Fc fused to the N-terminus of the broken intein Npu DnaE

The N-terminal gene of Npu DnaE and the Fc fragment of human IgG were cloned by the primers in Table 2, and amplified by PrimalStar Max of TaKaRa. The PCR conditions were 94 ° C for 10 s, 55 ° C for 10 s, 72 ° C for 10 s, 30 cycles. The obtained fragment was subjected to agarose gel electrophoresis, and the amplified fragment was synthesized by overlapping PCR in the order of Npu DnaE N-terminal-Fc fragment. The PCR conditions were 94 ° C for 10 s, 55 ° C for 10 s, 72 ° C for 10 s, 30 This gene fragment was circulated, treated with BamHI and NotI, and ligated with pET-22b(+) carrying dsscFv CD22, also treated with BamHI and NotI, with the fusion gene sequence dsscFv CD22-Nn-Fc.

The ligation product was transformed into E. coli DH5α competent cells, and the transformed cells were plated on an agar plate containing 50 μg/mL ampicillin overnight. The monoclonal clones grown on the plate were picked and shaken overnight in 5 mL of LB medium containing 50 μg/mL ampicillin, and the plasmid was extracted and sequenced. The sequencing results showed that the dsscFv CD22-Nn-Fc sequence constructed correctly was correct. . 2 is a schematic diagram showing the results of the fusion protein dsscFv CD22-Nn-Fc purified by a Ni-Sepharose column by SDS-PAGE in Example 1 of the method for preparing an immunotoxin using an intein.

Table 2

4. Expression and purification of fusion protein dsscFv-Nn-Fc

The correctly sequenced plasmid was transformed into the competent state of E. coli expression strain BL21, and the single bacteria were cultured overnight at 37 ° C. The single colony was cultured in 5 ml of LB medium containing 50 μg/mL ampicillin at 37 ° C, 180 rpm, and cultured overnight.

5 ml of the culture solution was added to 500 ml of fresh LB medium containing 50 μg/mL ampicillin, and cultured at 37 ° C until the OD600 was 0.6-0.8. IPTG was added to a final concentration of 0.02 mM, induced at 25 ° C for 16 h, and centrifuged at 4000 rpm for 10 min. Bacteria.

The collected cells were resuspended in binding buffer (20 mM PBS, 500 mM NaCl, 20 mM imidazole, pH 7.5, 20 ml of binding buffer per 1 g of the cells), and the cells were disrupted by a high-pressure homogenizer, and centrifuged at 12,000 rpm for 30 min at 4 ° C. The supernatant was collected, and the supernatant was filtered with 0.45 μm, and was purified by Ni ²⁺ NTA.

The Ni ²⁺ gravity column was packed, the volume of the bed was 1 ml, washed with 5 column volumes of water, and then equilibrated with 10 column volume of binding buffer (20 mM PBS, 500 mM NaCl, 20 mM imidazole, pH 7.5), and the collected supernatant was applied. Column, flow rate 1 ml/min, after completion of the upper column, rinse with 5 column volumes of Binding Buffer, followed by elution buffer containing 60 mM imidazole (10 mM PBS, 500 mM NaCl, 60 mM imidazole, pH 7.5) in 10 column volumes The non-specific binding protein was washed, and then eluted with 10 column volumes of 150 mM imidazole in elution buffer (20 mM PBS, 500 mM NaCl, 150 mM imidazole, pH 7.5), collected in a tube, 1 ml per tube, and eluted by SDS-PAGE. In the case, the target protein was combined to obtain the fusion protein dsscFv-Nn-Fc.

5. Preparation of dsscFv CD22-PE38KDEL by reverse splicing of Npu DnaE

The fusion polypeptide Nc-PE38KDEL obtained in the step 2 was mixed with the fusion polypeptide dsscFv CD22-Nn-Fc obtained in the step 4 at a molar ratio of 1:1, and DTT was added at a final concentration of 1 mM, and incubated at 25 ° C for 60 min to obtain a sample. SDS-PAGE and Western Blot assays were performed. Figure 3 is a schematic diagram showing the results of trans-splicing of dsscFv CD22-Nn-Fc and Nc-PE38KDEL to dsscFv CD22-PE38KDEL by Western Blot in Example 1 of the method for preparing an immunotoxin using intein.

6. Separation and purification of dsscFv CD22-PE38KDEL produced by Npu DnaE trans-splicing

The dsscFv CD22-PE38KDEL was purified by removing the fusion polypeptide in step 5 for the trans-splicing reaction and the by-product produced by trans-splicing using Ni ²⁺ NTA.

The Ni ²⁺ gravity column was packed, the volume of the bed was 1 ml, washed with 5 column volumes of water, and then equilibrated with 10 column volume of binding buffer (20 mM PBS, 500 mM NaCl, 20 mM imidazole, pH 7.5), and the reaction system obtained in the step 5 was used. The upper column was flowed at 1 ml/min, and the flow was collected. After the upper column was completed, it was rinsed with 5 column volumes of elution buffer (20 mM PBS, 500 mM NaCl, 40 mM imidazole, pH 7.5) containing 40 mM imidazole, followed by 10 The column volume was washed with 150 mM imidazole elution buffer (20 mM PBS, 500 mM NaCl, 150 mM imidazole, pH 7.5), and the collected protein samples were detected by SDS-PAGE, and frozen and at -20 ° C or -80 ° C as needed. Preservation, or for higher purity purification, such as ion exchange chromatography, hydrophobic chromatography, and size exclusion chromatography.

Example 2. Preparation of immunotoxin Her Fab-PE38KDEL by reverse splicing function of Npu DnaE

1. Construction of Her VH-CH1 expression vector pCEP4-Her VH-CH1-Nn fused to the N-terminus of the broken intein Npu DnaE

The gene encoding the heavy chain VH-CH1 part of Herceptin and the N-terminal gene of Npu DnaE were cloned by the primers in Table 3, and amplified by PrimalStar Max of TaKaRa. The PCR conditions were 94 ° C for 10 s, 55 ° C for 10 s, and 72 ° C for 10 s. After 30 cycles, the obtained fragment was subjected to agarose gel electrophoresis, and the gene encoding the Herceptin heavy chain VH-CH1 portion and the N-terminus of the N-terminal gene Npu DnaE encoding Npu DnaE were synthesized in the C-terminus of the fusion polypeptide by overlapping PCR. The PCR conditions were 94 ° C for 10 s, 55 ° C for 10 s, 72 ° C for 10 s, 30 cycles. The gene fragment was treated with HindIII and BamHI, and ligated with HindIII and BamHI-treated pCEP4. The plasmid structure is shown in Figure 7. .

The ligation product was transformed into E. coli DH5α competent cells, and the transformed cells were plated on an agar plate containing 50 μg/mL ampicillin overnight. The monoclonal clones grown on the plate were picked and shaken overnight in 5 mL of LB medium containing 50 μg/mL ampicillin, and the plasmid was extracted and sequenced. The sequencing results indicated that the constructed Her VH-CH1-Nn sequence was correct. .

table 3

2. Construction of the expression vector of Herceptin light chain pCEP4-Her LC

The genes encoding the Herceptin light chain were cloned by the primers in Table 4, and amplified by PrimalStar Max of TaKaRa. The PCR conditions were 94 ° C for 10 s, 55 ° C for 10 s, 72 ° C for 10 s, 30 cycles, and the obtained fragment agarose was coagulated. After gel electrophoresis, it was treated with HindIII and BamHI, and ligated with pCEP4 which was also treated with HindIII and BamHI. The plasmid structure is shown in Fig. 7.

The ligation product was transformed into E. coli DH5α competent cells, and the transformed cells were plated on an agar plate containing 50 μg/mL ampicillin overnight. The monoclonal clones on the plate were picked and cultured overnight in 5 mL of LB medium containing 50 μg/mL ampicillin, and the plasmid was extracted and sequenced. The sequencing results indicated that the constructed Her LC sequence was correct.

Table 4

3. Expression and purification of fusion polypeptide Her Fab-Nn

In this example, the fusion polypeptide Her Fab-Nn was expressed using a transient expression system in the HEK293-E system.

HEK293-E cells (human embryos expressing Epstein-Barr virus nuclear antigen) were cultured with SFM4HEK293 medium (HyClone) and Gibco Freestyle 293 medium (Gibco) in a ratio of 1:1 with 100 μg/ml geneticin (Gibco). Renal cell line 293; American Type Culture Center, accession number ATCC #CRL-10852, Lot. 959218), the cells were diluted to 1.5-2.5 × 10 ⁶ cells/ml with fresh medium one day before transfection, at 37 ° C , 120 rpm, 5% CO2 culture, to be transfected the next day.

On the day of transfection, DNA was diluted to 50 g/μL with Gibco Freestyle 293 medium in an amount equal to 0.5 μg of DNA per 106 cells in equal mass ratios of pCEP4-Her VH-CH1-Nn and pCEP4-Her LC. : PEI = 1:3 was added to the mixed DNA and incubated for 20 min at room temperature for use. The cells were collected by centrifugation at 1000 rpm for 5 min, and the cells were washed once with Gibco Freestyle 293 medium, centrifuged at 1000 rpm for 5 min, and resuspended in 150 ml Gibco Freestyle 293 medium to a cell density of 4×10 ⁶ cells/ml. 1L shake flask (Coming).

The incubated DNA-PEI complex was added to the cells and transfected at 37 ° C, 110 rpm, 5% CO 2 for 4 hours, followed by An equal volume of pre-warmed SFX4HEK293 medium was added, and 100 μg/ml geneticin (Gibco) was added to continue the culture at 37 ° C, 130 rpm, 5% CO 2 for 10 days. The supernatant was directly collected for purification or the supernatant was collected and stored at -80 ° C for cryopreservation.

The collected supernatant was mixed 1:1 with PBS (20 mM PBS, 150 mM NaCl, pH 6.8-7.4), and applied to a Protein L (protein L) affinity column equilibrated with PBS in advance, and the loading was completed 5 times. The column volume was washed with PBS, washed with 100 mM citrate buffer at pH 5.0 to remove the components, and the antibody was slowly eluted with 100 mM citric acid at pH 3.0 and immediately neutralized with 1 M tris-Hcl buffer at pH 9.0. The eluted sample to the point.

A small sample was taken for SDS-PAGG analysis. The assembled Her Fab-Nn appeared in the non-reduced sample at around 60KD, and the 35KD VH+CH1+Nn chain and the 25KD light chain appeared in the reduced sample. Figure 4 is a diagram showing the results of the fusion protein Fab-Nn purified by Protein L chromatography column by SDS-PAGE in Example 2 of the method for preparing an immunotoxin using inteins of the present invention.

Samples containing the protein of interest were pooled for subsequent intein-mediated in vitro splicing. If necessary, concentrate using a MILLIPORE Amicon Ultra (30 MWCO) ultrafiltration centrifuge tube, freeze and store at -20 ° C or -80 ° C.

4. Preparation of Her Fab-PE38KDEL by Npu DnaE trans-splicing

The fusion polypeptide Nc-PE38KDEL obtained in the step 2 of the first embodiment and the fusion polypeptide Her Fab-Nn obtained in the step 3 were mixed at a molar ratio of 1:1, and DTT was added at a final concentration of 1 mM, and the temperature was maintained at 25 ° C. At 60 min, samples were taken for SDS-PAGE and Western Blot. Figure 5 is a schematic diagram showing the results of Fb-Nn and Nc-PE38KDEL trans-splicing to generate Fab-PE38KDEL by SDS-PAGE in Example 2 of the method for preparing an immunotoxin using inteins of the present invention.

5. Purification of Her Fab-PE38KDEL by reverse splicing with Npu DnaE

Her Fab-PE38KDEL was purified by using Ni ²⁺ NTA to capture the fusion polypeptide in step 4 in which no trans-splicing reaction occurred and the by-product produced by trans-splicing.

The Ni ²⁺ gravity column was packed, the volume of the bed was 1 ml, washed with 5 column volumes of water, and then equilibrated with 10 column volume of binding buffer (20 mM PBS, 500 mM NaCl, 20 mM imidazole, pH 7.5), and the reaction system obtained in the step 4 was obtained. The upper column was flowed at 1 ml/min, and the flow was collected. After the upper column was completed, it was rinsed with 5 column volumes of 40 mM imidazole elution buffer (20 mM PBS, 500 mM NaCl, 40 mM imidazole, pH 7.5) to collect the rinsing solution. Then, rinse with 10 column volumes of 150 mM imidazole elution buffer (20 mM PBS, 500 mM NaCl, 150 mM imidazole, pH 7.5), and collect the collected protein samples by SDS-PAGE, and freeze and at -20 °C as needed. It can be stored at -80 ° C or used for higher purity purification, such as ion exchange chromatography, hydrophobic chromatography, and size exclusion chromatography.

Example 3, using the trans-splicing function of Npu DnaE to prepare the immunotoxin Herceptin-PE38KDEL

1. Construction of the Her HC expression vector pCEP4-Her HC-Nn fused to the N-terminus of the broken intein Npu DnaE

Using the primers in Table 5 below, the synthetic Herceptin heavy chain nucleic acid molecule encoding the signal peptide gene was used as a template to clone the gene encoding the Herceptin heavy chain using the primers in the table, and the Npu DnaE-containing nucleic acid molecule was used as a template to clone N of Npu DnaE. The terminal gene was amplified by PrimalStar Max of TaKaRa, and the PCR conditions were 94 ° C for 10 s, 55 ° C for 10 s, 72 ° C for 10 s, 30 cycles, and the obtained fragment was subjected to agarose gel electrophoresis recovery, and the overlap encoding was used to encode Herceptin. The N-terminus of the N-terminal gene Npu DnaE encoding Npu DnaE was synthesized at the C-terminus of the fusion polypeptide. The PCR conditions were 94 ° C for 10 s, 55 ° C for 10 s, 72 ° C for 10 s, 30 cycles, and the gene fragment was used for Hin dIII. It was treated with BamHI and ligated with pCEP4 which was also treated with HindIII and BamHI. The plasmid structure is shown in Fig. 7.

The ligation product was transformed into E. coli DH5α competent cells, and the transformed cells were plated on an agar plate containing 50 μg/mL ampicillin overnight. The monoclonal clones grown on the plate were picked and shaken overnight in 5 mL of LB medium containing 50 μg/mL ampicillin, and the plasmid was extracted and sequenced. The sequencing results indicated that the constructed Her HC-Nn sequence was correct.

table 5

2. Expression and purification of fusion polypeptide Herceptin-Nn

In this example, the fusion peptide Herceptin-Nn was expressed using a transient expression system in the HEK293-E system.

HEK293-E cells (human embryos expressing Epstein-Barr virus nuclear antigen) were cultured in a ratio of 1:1 with SFX4HEK293 medium (HyClone) and Gibco Freestyle 293 medium (Gibco) supplemented with 100 μg/ml geneticin (Gibco). Renal cell line 293; American Type Culture Center, accession number ATCC #CRL-10852, Lot. 959218), the cells were diluted to 1.5-2.5 × 10 ⁶ cells/ml with fresh medium one day before transfection, at 37 ° C Cultured at 120 rpm, 5% CO ₂ until the next day.

On the day of transfection, the recombinant plasmid pCEP4-Her HC-Nn and the Herceptin light chain expression vector pCEP4-Her LC constructed in step 2 of Example 3 were mixed with 0.5 μg of DNA per 10 ⁶ cells, and cultured with Gibco Freestyle 293. The DNA was diluted to (40 ng/μL), DNA: PEI = 1:3, and the mixed DNA was added and incubated for 20 min at room temperature for use. The cells were collected by centrifugation at 1000 rpm for 5 min, the cells were washed once in Gibco Freestyle 293 medium, centrifuged at 1000 rpm for 5 min, and the cells were resuspended in 150 ml Gibco Freestyle 293 medium to a cell density of 4×10 ⁶ cells/ml. 1L shake flask (Coming).

The incubated DNA-PEI complex was added to the cells, transfected at 37 ° C, 110 rpm, 5% CO 2 for 4 hours, followed by the addition of an equal volume of pre-warmed SFX 4 HEK293 medium, and 100 μg/ml geneticin (Gibco) was added. Incubate for 10 days at 37 ° C, 130 rpm, 5% CO 2 .

The supernatant was directly collected for purification or the supernatant was collected and stored at -80 ° C for cryopreservation.

The collected supernatant was mixed 1:1 with PBS (20 mM PBS, 150 mM NaCl, pH 6.8-7.4), and applied to a Protein A affinity column equilibrated with PBS in advance, and the loading was 10 times. The column volume was washed with PBS, the antibody was eluted with 100 mM citrate buffer at pH 3.0, and the collected eluted samples were immediately neutralized with 1 M Tris-Hcl buffer at pH 9.0. A small sample was taken for SDS-PAGG analysis, and the assembled Herceptin-Nn appeared at about 170 kD in the non-reduced sample, and a Herk-Nn chain of about 70 kD and a light chain of 25 KD appeared in the reduced sample. Samples containing the protein of interest were pooled for subsequent intein-mediated in vitro splicing. If necessary, concentrate using a MILLIPORE Amicon Ultra (30 MWCO) ultrafiltration centrifuge tube, freeze and store at -20 ° C or -80 ° C.

3. Preparation of Herceptin-PE38KDEL by Npu DnaE trans-splicing

The fusion polypeptide Nc-PE38KDEL obtained in the step 2 of the first embodiment and the fusion polypeptide Herceptin-Nn obtained in the step 3 were mixed at a molar ratio of 1:1, and DTT was added at a final concentration of 1 mM, and incubated at 25 ° C for 60 min. The samples were taken for SDS-PAGE and Western Blot. 6 is a schematic diagram showing the results of the method for preparing an immunotoxin by using an intein according to the third embodiment of the method for detecting Herceptin-PE38KDEL by trans-splicing of Herceptin-Nn and Nc-PE38KDEL by Western Blot.

4. Separation and purification of Herceptin-PE38KDEL produced by Npu DnaE trans-splicing

Herceptin-PE38KDEL was purified by using Ni ²⁺ NTA to capture the fusion polypeptide in step 3 in which no trans-splicing reaction occurred and the by-product produced by trans-splicing. The Ni ²⁺ gravity column was packed, the volume of the bed was 1 ml, washed with 5 column volumes of water, and then equilibrated with 10 column volume of binding buffer (20 mM PBS, 500 mM NaCl, 20 mM imidazole, pH 7.5), and the reaction system obtained in the step 3 was obtained. The upper column was flowed at 1 ml/min, and the flow was collected. After the upper column was completed, it was rinsed with 5 column volumes of 40 mM imidazole elution buffer (20 mM PBS, 500 mM NaCl, 40 mM imidazole, pH 7.5) to collect the rinsing solution. Then, rinse with 10 column volumes of 150 mM imidazole elution buffer (20 mM PBS, 500 mM NaCl, 150 mM imidazole, pH 7.5), and collect the collected protein samples by SDS-PAGE, and freeze and at -20 °C as needed. It can be stored at -80 ° C or used for higher purity purification, such as ion exchange chromatography, hydrophobic chromatography, and size exclusion chromatography.

It can be seen that the method of the present invention overcomes the deficiencies in the traditional strategies for the production of immunotoxins, and achieves many deficiencies in the technical methods for improving the traditional production of immunotoxins, such as the need to add chemical reagents in the chemical coupling method. The product is heterogeneous due to the large number of modification sites in the targeting polypeptide moiety, and the chemically coupled bond is easily broken to cause toxic leakage. However, in the strategy of directly expressing the immunotoxin fusion protein, if the prokaryotic expression system is used, the purpose is Proteins often exist as inclusion bodies, and the renaturation efficiency is low and the cumbersome steps are complicated. If eukaryotic expression systems are used, the expression of immunotoxins may be limited by their natural toxicity to host cells. The method of the invention improves the deficiencies of the prior art: 1. The method of the invention only needs to prepare different targeting part polypeptides and toxic partial polypeptides, which can be combined to produce different toxic mechanisms for different targets and different targets. The immunotoxin has significant diversity and flexibility; 2. In the method of the present invention, the targeting part polypeptide and the toxic part polypeptide can be separately expressed in a suitable host cell, such as a special folding environment, especially an advanced translation. Post-modification can be expressed in mammalian cells, while polypeptides without much modification requirements can be expressed in E. coli, and the expression of the polypeptide of interest in a suitable expression system can obtain higher yield, yield and purity; 3. In the method of the present invention, the linkage between the targeting moiety polypeptide and the toxin partial polypeptide is site-specific, does not produce by-products, and the obtained product product is highly homogenous; 4. In the method of the present invention, the targeting moiety polypeptide And the self-splicing of the toxin partial polypeptide via the intein, which is linked by peptide bonds, compared to The coupling method and the like have good stability; 5. In the method of the invention, the self-splicing reaction condition is mild, the reaction is efficient, and it is easy to integrate and amplify with other processes; the reaction process does not need to add toxic and harmful substances.

The specific embodiments of the present invention have been described above. It is to be understood that the invention is not limited to the specific embodiments described above, and various modifications and changes may be made by those skilled in the art without departing from the scope of the invention.

Claims

A method for synthesizing a pharmaceutical recombinant protein based on an intein, characterized in that the method comprises the following steps:

Step one: constructing a vector DNA fragment expressing a polypeptide having a guiding function in an immunotoxin and a polypeptide having toxic function, and connecting the vector DNA fragment of the two polypeptides to the N-terminus or the C-terminus of the intein to form a fusion expression. a vector; wherein the polypeptide expressed by the fusion expression vector containing the N-terminus of the intein and the fusion expression vector containing the C-terminus of the intein is required to be paired so that the trans-splicing reaction can occur;

Step 2, the fusion expression vector described in the first step is separately expressed in a suitable host cell to obtain a fusion polypeptide A fused to the N-terminus of the intein and a fusion polypeptide B fused to the C-terminus of the intein;

In the third step, the fusion polypeptide A and the fusion polypeptide B are taken, mixed, and the polypeptide which induces the guiding function of the immunotoxin and the polypeptide part having the toxic function are trans-spliced under the trans-splicing condition corresponding to the intein to obtain an immunotoxin.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in step 1, the intein is an artificially-cleaved intein, or a naturally-cleaved intein, or has a splicing Functional intein mutants.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in the first step, the intein is Ssp DnaB, Ssp DnaE or Npu DnaE.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in the first step, the N-terminus of the N-terminus of the intein or the N-terminus of the C-terminus is further fused with a functional polypeptide.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein the functional polypeptide is an MBP or ChBD tagged protein.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in the first step, the fusion expression vector is configured to direct the functional polypeptide-intein N-terminus or intein C-terminally toxic functional polypeptide, or toxic functional polypeptide-intein N-terminal, or intein C-terminal-directed functional polypeptide, or directed functional polypeptide-intein N-terminal-functional polypeptide, or functional polypeptide-intein C-terminal toxic functional polypeptide, or toxic functional polypeptide-intein N-terminal functional polypeptide, functional polypeptide-intein C-terminal-directed functional polypeptide.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in the first step, the polypeptide having a guiding function is an antibody, a single-chain antibody scFv, a disulfide-stabilized antibody dsFv, Disulfide-linked single-chain antibody dsscFv, Fab antibody, cytokine, or growth factor.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in the first step, the toxic functional polypeptide is a RIP type toxin; specifically ricin, ADP ribosylation An immunotoxin, a fusion protein of diphtheria toxin, or a Pseudomonas aeruginosa exotoxin moiety, or a mutant having toxin biological activity.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in the second step, the host cell is a prokaryotic cell or a eukaryotic cell.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein the host cell is Escherichia coli, Bacillus subtilis, high-yield yeast cells, insect cells, plant cells, mammalian cells. , yeast, Chinese hamster ovary cells, or human kidney blasts.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in the third step, the step of separating and purifying to obtain an immunotoxin is further included.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein the separation and purification method is an affinity chromatography method, an ion exchange chromatography method, or a hydrophobic interaction chromatography method.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein in the third step, the trans-splicing condition is a condition capable of triggering the specified intein to complete self-splicing.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein when the intein is Npu DnaE, the trans-splicing conditions are: 0-55 ° C, pH 3-11, The nucleophilic reagent was contacted for a period of 1 s to 60 h with a final concentration of 0.05 mM to 100 mM.
The method for synthesizing an intein-based pharmaceutical recombinant protein according to claim 1, wherein the nucleophilic reagent is DTT, DTE, CYSTEINE, TCEP, or MESNA nucleophilic reagent.