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Foreign protein soluble expression plasmid, preparation method thereof and application method thereof

A technology of exogenous proteins and application methods, applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, etc., can solve problems such as the effect of co-expression of folding auxiliary proteins

Inactive Publication Date: 2011-01-12
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, temperature may also have an important impact on the effect of co-expression of folding accessory proteins
For example, co-expression of preS2-S'-β-galactosidase with DnaK and DnaJ chaperones can increase the expression level of soluble proteins in the range of 30 to 42 °C, while co-expression of GroEL and GroES chaperones can only increase the expression level of soluble proteins at 30 °C. Better results can be obtained under the condition of ℃

Method used

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  • Foreign protein soluble expression plasmid, preparation method thereof and application method thereof
  • Foreign protein soluble expression plasmid, preparation method thereof and application method thereof
  • Foreign protein soluble expression plasmid, preparation method thereof and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Schematic diagram of pET30e expression vector in Escherichia coli and SDS-PAGE analysis of expression of molecular chaperone FaeE in Escherichia coli

[0045] The molecular chaperone FaeE coding sequence was cloned into the commercialized expression vector pET30a through two restriction enzymes Nde I and Bam HI, so that the gene was located under the control of the T7 promoter and the lac operator. The E. coli ribosome binding site is added to the 5' end, followed by multiple cloning sites that can be used to clone foreign protein genes, including Bam HI, EcoR I, Sac I, Hind III, Not I, Xho I, followed by the multiple cloning site is a 6-histidine coding sequence. Foreign genes are terminated by the T7 terminator. Carrier structure such as figure 1 with figure 2 .

[0046] The pET30a and obtained pET30e E. coli expression vectors were transformed into E. coli expression strain BL21 (DE3), and the single colonies of recombinant E. coli pET30a / BL21 (DE3) and pET30e / B...

Embodiment 2

[0048] SDS-PAGE and Western blot analysis of pET30CTB and pET30eCTB expressed in Escherichia coli

[0049] The coding sequence of the CTB coding sequence was cloned into pET30e treated with the same digestion by Bam HI and Xho I to obtain the expression vector pET30eCTB. The vector structure is as follows: image 3 .

[0050] The DNA sequence of the vector pET30eCTB plasmid is shown in Seq ID No.2, which includes the coding sequence of the molecular chaperone FaeE and the coding sequence of the cholera toxin B subunit CTB, and the cholera toxin B subunit (CTB) coding sequence It was amplified from classical Vibrio cholerae (CVC) genomic DNA as a template.

[0051] Transform pET30CTB and pET30eCTB into Escherichia coli expression strain BL21(DE3) to obtain recombinant Escherichia coli pET30CTB / BL21(DE3) and pET30eCTB / BL21(DE3). The methods of bacterial culture, protein induction and treatment are the same as in Example 1. The protein is separated by SDS-PAGE. After separation...

Embodiment 3

[0054] SDS-PAGE and Western blot analysis of pET30PRX and pET30EPRX expressed in Escherichia coli

[0055] The coding sequence of PRX was cloned into pET30e treated with the same enzyme digestion by Bam HI and Xho I to obtain the expression vector pET30ePRX, the vector structure is as follows Figure 4 .

[0056] The DNA sequence of the vector pET30ePRX plasmid is shown in Seq ID No.3, comprising the coding sequence of molecular chaperone FaeE and the coding sequence of rice endogenous peroxidase, and the rice endogenous peroxidase (PRX) The gene sequence was amplified using rice genomic DNA as a template.

[0057] Transform pET30PRX and pET30EPRX into Escherichia coli expression strain BL21(DE3) to obtain recombinant Escherichia coli pET30PRX / BL21(DE3) and pET30ePRX / BL21(DE3). The methods for bacterial culture, protein induction and treatment are the same as in Example 1, and the Western blot analysis method is the same as in Example 2. SDS-PAGE results ( Image 6 A) show...

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Abstract

The invention discloses a foreign protein soluble expression plasmid, a preparation method thereof and application method thereof, which belong to the technical field of bioengineering. The plasmid is pET30E, wherein the DNA sequence of the plasmid is presented by Seq ID No.1. An amplified product obtained by performing PCR amplification on a plasmid template and a primer group is connected with a pMD18-t vector; an Escherichia coli clone is obtained by transforming Escherichia coli DH5 alpha; and a product obtained by performing enzyme cutting connection on the Escherichia coli clone serving as a substrate is used for transforming the Escherichia coli DH5 alpha to form soluble expression plasmid molecules. The application method comprises the following steps that: the obtained expression plasmid molecules are used to transform Escherichia coli BL21 (DE3) competent cells through thermal activation to form a recombinant Escherichia coli colony; and after a bacteroid cell is obtained through inoculation culture, the soluble expression of the expression plasmid molecule is improved. The plasmid of the invention has promotion effect on assistance of the soluble expression of foreign proteins in Escherichia coli and has a great significance for further analysis of the functions of the proteins.

Description

technical field [0001] The present invention relates to a plasmid in the technical field of bioengineering and its preparation and application method, in particular to a plasmid for improving the soluble expression of foreign protein in E. coli by utilizing the FaeE gene of enterotoxigenic Escherichia coli and its preparation and application method . Background technique [0002] Escherichia coli has the characteristics of thorough understanding of genetic traits, fast growth, economical culture, high expression level, and many plasmids and hosts to be selected. It has become the preferred expression system in the field of genetic engineering technology. However, foreign proteins are often easily degraded by host proteases or form inclusion bodies while obtaining high-level expression. At present, there are many studies on protein renaturation in vitro at home and abroad, but the process is often time-consuming, laborious, and uneconomical. Therefore, exploring the soluble ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/64C12N15/67
CPCC12N15/72C12N15/67
Inventor 张大兵申慧峰梁婉琪袁政
Owner SHANGHAI JIAO TONG UNIV
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