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Plasmid for heterologous protein solubility expression and preparation and application method thereof

A technology of exogenous protein and application method, which is applied to the plasmid for soluble expression of exogenous protein and the fields of its preparation and application, and can solve the problems of the effect of co-expression folding auxiliary protein and the like

Inactive Publication Date: 2012-07-04
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Plasmid for heterologous protein solubility expression and preparation and application method thereof
  • Plasmid for heterologous protein solubility expression and preparation and application method thereof
  • Plasmid for heterologous protein solubility expression and preparation and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Schematic diagram of pET30e expression vector in Escherichia coli and SDS-PAGE analysis of expression of molecular chaperone FaeE in Escherichia coli

[0046] 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 and figure 2 .

[0047] Transform pET30a and the obtained pET30e E. coli expression vector into human E. coli expression strain BL21(DE3), and single colony of recombinant E. coli pET30a / BL21(DE3) and pET30e / BL21(DE3) in...

Embodiment 2

[0049] SDS-PAGE and Western blot analysis of pET30CTB and pET30eCTB expressed in Escherichia coli.

[0050] 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 .

[0051] 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.

[0052] 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 separatio...

Embodiment 3

[0055] SDS-PAGE and Western blot analysis of pET30PRX and pET30EPRX expressed in Escherichia coli

[0056] 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 .

[0057] 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.

[0058] 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 ( Figure 6 A) sho...

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Abstract

The invention relates to a plasmid for heterologous protein solubility expression and a preparation and application method thereof. The plasmid is PET30e, and a deoxyribonucleic acid (DNA) sequence of the plasmid is described as Seq ID No.1. Amplification product obtained by conducting polymerase chain reaction (PCR) amplification on a plasmid template and primers is connected with a pMD18-T vector, clone of escherichia coli is obtained by transforming escherichia coli DH5 alpha and serves as a substrate to conduct restriction enzyme digestion and connection to obtain a product which transforms the escherichia coli DH5 alpha to obtain solubility expression plasmid molecules. The application method includes that the obtained expression plasmid molecules transform escherichia coli BL21 (DE3) competent cells to obtain a recombination escherichia coli bacterial colony in heat shock method, somatic cells are obtained through inoculation cultivation to obtain improvement of solubility expression. The plasmid for the heterologous protein solubility expression and the preparation and application method of the plasmid can improve solubility expression of the heterologous protein in the escherichia coli and have important meaning for further analyzing functions of the protein.

Description

[0001] The patent application for this invention is China Invention Patent Application No. 201010266983.5, the application date is August 31, 2010, the applicant is "Shanghai Jiaotong University", and the name of the invention is "Plasmid for soluble expression of exogenous protein and its preparation and application method". case application. technical field [0002] 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 [0003] 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 en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/67C12N15/64C12R1/19
Inventor 张大兵梁婉琪袁政申慧峰
Owner SHANGHAI JIAO TONG UNIV
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