Soluble expression system and application thereof in soluble expression of protein

An expression system and soluble technology, which is applied in the application field of soluble expression system and protein soluble expression, can solve the problems of complex operation and low renaturation rate, and achieve the effect of reducing costs

Pending Publication Date: 2022-04-12
江苏万邦医药科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, many exogenous proteins expressed by Escherichia coli, including porcine carboxypeptidase B (proCPB), exist in the cells in the form of inclusion bodies, and subsequent renaturation of the products is required. The operation is complicated and the renaturation rate Low (Zhang Xiaoyan, Zhang Huiyong, Fan Hao, Li Taiming, Liu Jingjing, Cao Rongyue. Expression and renaturation of recombinant porcine procarboxypeptidase B in Escherichia coli. Pharmaceutical Biotechnology, 2010,17(1):39–44)

Method used

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  • Soluble expression system and application thereof in soluble expression of protein
  • Soluble expression system and application thereof in soluble expression of protein
  • Soluble expression system and application thereof in soluble expression of protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Embodiment 1, plasmid construction

[0128] 1. Construction of plasmid p15ARA

[0129] araC-P BAD The sequence was derived from the genome sequence of E. coli BL21 (DE3) (NCBI VERSION: CP001509.3).

[0130] 1. Using the BL21(DE3) genome as a template and PARA-5 and PARA-3 as primers, about 1.7kb of PCR amplification containing araC gene, P BAD Promoter and DNA fragment ARA of partial araB gene sequence.

[0131] 2. Using plasmid pACYCDuet-1 as template, ACYC-5 and ACYC-3 as primers, PCR amplification containing Cm R and the DNA backbone ACYC of p15Aori (about 2.2 kb).

[0132] 3. In vitro recombination of backbone ACYC and fragment ARA to construct plasmid p15ARA (structural schematic diagram as shown in figure 1 shown). All in vitro recombination operations were completed using the recombination cloning kit of Nanjing Novizan Biotechnology Co., Ltd.

[0133] 4. Pick a single colony and use P15AC and ACYC-33 as primers for PCR identification. About 1.4kb is posit...

Embodiment 2

[0328] Embodiment 2, the construction of bacterial strain

[0329] 1. Construction of expression strains of sulfhydryl oxidase and protein disulfide bond isomerase

[0330] 1. The plasmid p15ARA1 was transformed into the strain BL21(DE3), and the strain BL21(DE3) / p15ARA1 was obtained, which was named WF1. This strain expresses ERV1 and hPDI.

[0331] 2. The plasmid p15ARA2 was transformed into the strain BL21(DE3), and the strain BL21(DE3) / p15ARA2 was obtained, which was named WF2. This strain expresses ERV1 and sPDI.

[0332]3. The plasmid p15ARA3 was transformed into the strain BL21(DE3), and the strain BL21(DE3) / p15ARA3 was obtained, which was named WF3. This strain expresses ERV1 and DsbC.

[0333] 4. The plasmid p15T1 was transformed into the strain BL21(DE3), and the strain BL21(DE3) / p15T1 was obtained, which was named WF4. This strain expresses ERV1 and hPDI.

[0334] 5. The plasmid p15T2 was transformed into the strain BL21(DE3), and the strain BL21(DE3) / p15T2 wa...

Embodiment 3

[0377] Example 3, N-terminal tag optimization strategy to achieve soluble expression of sulfhydryl oxidase and protein disulfide bond isomerase

[0378] Culture strains BL21(DE3), WF1, WF2 and WF3, when OD 600nm Adding L-arabinose at 0.3–0.4 induces the expression of corresponding sulfhydryl oxidase (ERV1) and protein disulfide bond isomerase (hPDI, sPDI or DsbC), and the SDS-PAGE electrophoresis results of soluble protein and inclusion body protein are as follows Figure 43 shown. The protein molecular weights of ERV1, hPDI, sPDI and DsbC are about 21.70 kDa, 52.05 kDa, 55.70 kDa and 23.51 kDa, respectively. Compared with the control BL21(DE3), there is no band of the target protein.

[0379] The promoters of the sulfhydryl oxidase and protein disulfide isomerase genes were replaced with stronger T7 promoters. Cultivate strains BL21(DE3), WF4, WF5 or WF6, add inducer IPTG, express corresponding sulfhydryl oxidase (ERV1) and protein disulfide bond isomerase (hPDI, sPDI or D...

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Abstract

The invention discloses a soluble expression system and application thereof in soluble expression of protein. The inventor screens protein disulfide bond isomerase from different sources, and constructs the protein disulfide bond isomerase and sulfydryl oxidase under the control of the same promoter. The expression of the two proteins is optimized through a strategy of adding a nitrogen terminal tag, and a soluble expression system is constructed. In escherichia coli, after co-expression of the system and target protein recombinant porcine carboxypeptidase B, soluble expression of the porcine carboxypeptidase B can be realized, and formation of inclusion bodies is greatly reduced. The soluble expression system can also be used for soluble expression of other target proteins, such as trypsinogen, and has universality of promoting soluble expression of the target proteins. The method has application prospects in the fields of recombinant protein production, new enzyme original screening and the like.

Description

technical field [0001] The invention belongs to the field of recombinant protein expression, more specifically, the invention relates to a soluble expression system, its construction and its application in the soluble expression of protein including recombinant porcine carboxypeptidase pro-B. Background technique [0002] Protease plays an important role in the fields of drug production and scientific research. Carboxypeptidase B (Carboxypeptidases B, CPB) is a protease containing metal zinc, which can hydrolyze arginine (Arg) or lysine at the C-terminus of protein or polypeptide substrates. (Lys), plays an important role in drug production, scientific research and diagnosis of acute pancreatitis (Francesc XAvilés, Josep Vendrell, Alicia Guasch, Miquel Coll, Robert Huber, Advances inmetallo-procarboxypeptidases. European Journal of Biochemistry, 1993, 211 (3):381–389; Lihui Deng, Lei Wang, Fengjiao Yong, Junjie Xiong, Tao Jin, Daniel De LaIglesia-Garcia, Shameena Bharucha, K...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/61C12N15/53C12N9/48C12N9/76C12R1/19
Inventor 文良柱方宏清韦炎龙窦俊
Owner 江苏万邦医药科技有限公司
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