Efficient method for producing Trx-hCTRP2

A high-efficiency, pmd20-t technology, applied in the field of high-efficiency production of Trx-hCTRP2, can solve the problems of inactivation of the target product, instability of hCTRP2 in yeast, inability to obtain full-length and uniform hCTRP2 protein, etc., and achieve the effect of good biological activity.

Inactive Publication Date: 2011-10-05
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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Problems solved by technology

Our research group used the yeast expression system to express it. Although hCTRP2 can be secreted and expressed in yeast, the expressed hCTRP2 is very unstable in yeast. hCTRP2 protein (see Figure 8 )
The expression product of Escherichia coli usually exists in the form of inclusion bodies, and the active form of hCTRP2 can only be obtained through complex operations of denaturation and renaturation, thereby reducing the yield of active protein. The inventors of the present application also use many other expression

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  • Efficient method for producing Trx-hCTRP2
  • Efficient method for producing Trx-hCTRP2
  • Efficient method for producing Trx-hCTRP2

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Embodiment Construction

[0028] In the present invention, the Escherichia coli expression strain BL21 (DE3), the vector amplification strain TOP10 and the expression vector pET32-a (+) were all purchased from Invritrogen Company of the United States.

[0029] The medium formula used is as follows:

[0030] 1) LB liquid medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, autoclave, store at room temperature;

[0031] 2) LB / Amp plate: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, agar powder 15g, after autoclaving, cool to below 70°C, add 1mL Ampicillin (100mg / ml), mix well and pour the plate , stored in the dark at 4°C;

[0032] 3) LB / Amp medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, after autoclaving, cool to below 70°C, add 1mL Ampicillin (100mg / ml), mix well, store at 4°C. LB liquid medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, autoclave, store at room temperature;

[0033] 4) 50×TAE agarose gel electrophoresis buffer: ...

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Abstract

The invention discloses an efficient method for producing Trx-hCTRP2. The method mainly comprises the following steps of: (1) cloning a human hCTRP2 gene; (2) constructing a prokaryotic expression vector by carrying out a double digestion on an expression vector pET32-a (+) and a plasmid hCTRP2/ pMD20-T with XhoI and BamH I, purifying for recovery, and utilizing a T4DNA ligase for connection to obtain a recombinant vector pET32/ hCTRP2; (3) conversing the recombinant vector into a escherichia coli host; (4) carrying out soluble induction expression; (5) purifying. According to the method of the invention, pET32 is employed as the vector and inducible expression conditions are optimized, so as to highly express a soluble hCTRP2 fusion protein. The method not only guarantees biological activities of the hCTRP2 fusion protein, but also enables efficient obtainment of a large amount of stabilized proteins.

Description

technical field [0001] The invention relates to the technology of applying recombinant DNA technology to produce genetically engineered protein medicine, in particular to a highly efficient production of thioredoxin (Trx)-hCTRP2 fusion protein with a histidine tag. technical background [0002] Adiponectin is a cytokine secreted by adipocytes, first discovered in 1995 by Scherer et al. in 3T3 adipocytes of mice. Studies have shown that it has the effects of increasing fatty acid oxidation, increasing glucose intake, improving insulin resistance, regulating inflammatory response of vascular endothelium, and is closely related to obesity, type 2 diabetes and cardiovascular disease. CTRPs (Clq-tumor necrosis factor-related protein) is a new type of protein family that is similar in structure and function to adiponectin. There are 12 cDNAs and proteins of this family (CTRP1-12) found in the NCBI database sequence. CTRPs proteins consist of four distinct domains: an N-terminal ...

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Application Information

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IPC IPC(8): C07K19/00C12N15/70C07K1/22C07K1/14C12R1/19
Inventor 吴东海李洪波金守光徐爱民李侍武
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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