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Method for preparing soluble HIV-1 integrase recombinant protein

A HIV-1, recombinant protein technology, applied in the direction of recombinant DNA technology, transferase, introduction of foreign genetic material using vectors, etc., can solve the problems of low yield, easy formation of inclusion bodies, poor solubility of target protein, etc., to increase the solubility , Strengthen the crushing effect, increase the effect of solubility and stability

Inactive Publication Date: 2013-09-04
BEIJING UNIV OF TECH
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Problems solved by technology

However, when using existing prokaryotic expression and purification methods to prepare integrase recombinant proteins, there are generally problems such as poor solubility of the target protein, easy formation of inclusion bodies, and low yield, which will affect the follow-up research to a certain extent.

Method used

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  • Method for preparing soluble HIV-1 integrase recombinant protein

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Embodiment Construction

[0015] The present invention will be further described below in conjunction with specific embodiment:

[0016] Prepare soluble HIV-1 integrase recombinant protein according to the following steps:

[0017] (1) Transform the integrase recombinant protein expression vector pWIN constructed on the basis of the pET-28a (+) vector into Escherichia coli BL21 (DE3) competent cells, pick positive clones and inoculate them in 5 ml containing 50 mg / L cannabinoid Mycin LB liquid medium was shaken at 37 °C for 14 h to obtain cultures.

[0018] (2) The culture in (1) was inoculated into 0.6 L expression medium at a volume ratio of 1%, and cultured on a shaking table at 37 °C until the OD600 value of the bacterial solution reached 0.8, and then added isopropyl-β-D-thio The final concentration of galactoside to isopropyl-β-D-thiogalactoside was 0.4 mM, and the culture was obtained by shaken culture at 37 °C for 4.5 h; the expression medium contained 25 g / L tryptone (purchased from Thermo F...

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Abstract

The invention discloses a method for preparing a soluble HIV-1 integrase recombinant protein, and belongs to the technical field of biology. The preparation method comprises the steps of induced expression, bacteria disruption, purification and the like. The adopted expression medium contains 15 to 35 g / L of tryptone, 10 to 20 g / L of yeast extract, 2 to 3 g / L of sodium chloride, 1 g / L of glucose, 10 to 15 g / L of glycerin, 2.05 g / L of disodium hydrogen phosphate, 1.27 g / L of sodium dihydrogen phosphate, 50 mg / L of magnesium sulfate, and 60 mg / L of kanamycin; and the adopted cracking buffer solution contains 20 mM of 4-hydroxyethyl piperazine ethanesulfonic acid, 1 M of sodium chloride, 2 mM of beta-mercaptoethanol, 0.3 mg / ml of lysozyme and 5 mM of imidazole, and the pH value of the buffer solution is 9.0. The expression medium can ensure normal growth of host bacteria and efficient expression of the objective protein; and the cracking buffer solution can enhance the disruption effect of bacteria, improve the solubility of the objective protein and finally improve the yield of the objective protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of soluble HIV-1 integrase recombinant protein. Background technique [0002] HIV-1 integrase is encoded by the HIV-1 pol gene, contains 288 amino acid residues, and has a molecular weight of about 32 kDa. Integrase catalyzes the integration of HIV-1 cDNA formed by reverse transcription into the genome of host cells, which is an essential link in the HIV-1 replication cycle. Since there are no functional analogues of integrase in human cells, inhibitors acting on integrase have little potential side effects on the human body, so integrase is considered to be an ideal target for anti-HIV-1 drug research. [0003] In the mechanism and drug research related to HIV-1 integrase, it is usually necessary to prepare recombinant integrase protein by means of prokaryotic expression and purification. However, when using existing prokaryotic expression and puri...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/70
Inventor 李春华李杉刘斌何红秋段彦华张小轶谭建军王存新
Owner BEIJING UNIV OF TECH
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