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Production method of gene recombinant protein Tat-hMsrA

A technology of trx-tat-hmsra and genetic recombination, which is applied in the biological field and can solve problems such as inactivity

Active Publication Date: 2019-10-29
WUHAN HUA PEPTIDE BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purified target protein obtained from the inclusion body has no activity after detection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The present embodiment provides a method for obtaining a positive Bacillus subtilis containing the Tat-hMsrA gene of interest, comprising the following steps:

[0032] S1, constructing the target gene containing signal peptide SacB gene, fusion protein Trx-Tat-hMsrA and enzyme cutting sites EcoR I and BamHI.

[0033] S2, the target gene and the shuttle vector pGJ148 were double-digested with EcoR I and BamH I respectively, after the gel was recovered, the ligation product was obtained by the action of T4 ligase, and the ligation product was transformed into Escherichia coli competent cells DH5α to extract the recombinant plasmid.

[0034] S3, the recombinant plasmid is electrotransformed into the host Bacillus subtilis, and the strain is cultured to obtain positive expression bacteria.

Embodiment 2

[0036] This embodiment provides a method for producing the recombinant protein Tat-hMsrA, comprising the following steps:

[0037] S1, culture the positive expression bacteria of Bacillus subtilis obtained in Example 1, the culture conditions are: LB medium containing neomycin with a final concentration of 15 μg / mL and chloramphenicol with a final concentration of 7 μg / mL, cultured at 37°C 12h.

[0038] S2, placing the positive Bacillus subtilis expression bacteria cultured in step S1 in LB medium containing 10% maltose for induction culture, and culturing for 2 days to obtain an expression product liquid containing Tat-hMsrA.

[0039] S3, centrifuge the Tat-hMsrA expression product solution obtained in step S2, discard the precipitated bacteria, add enterokinase digestion buffer to the supernatant for digestion, and obtain a digestion product solution.

[0040] S4, adding a phosphate buffer solution containing 0.7 mg / mL lysozyme, 2.0 mg / L glutamine and 0.1 mol / L zinc chlorid...

Embodiment 3

[0043] This embodiment provides a method for producing the recombinant protein Tat-hMsrA, comprising the following steps:

[0044] S1, the positive Bacillus subtilis expression bacteria obtained in Example 1 were cultured, and the culture conditions were: LB medium containing neomycin at a final concentration of 20 μg / mL and chloramphenicol at a final concentration of 3 μg / mL, cultured at 37°C for 12 hours .

[0045] S2, placing the positive Bacillus subtilis expression bacteria cultured in step S1 in LB medium containing 10% maltose for induction culture, and culturing for 2 days to obtain an expression product liquid containing Tat-hMsrA.

[0046] S3, centrifuge the Tat-hMsrA expression product solution obtained in step S2, discard the precipitated bacteria, add enterokinase digestion buffer to the supernatant for digestion, and obtain a digestion product solution.

[0047] S4, adding a phosphate buffer solution containing 1.0 mg / mL lysozyme, 1.0 mg / L glutamine and 0.5 mol / ...

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PUM

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Abstract

The invention relates to a production method of a gene recombinant protein Tat-hMsrA. The production method comprises the following steps that recombinant plasmid containing a fusion protein Trx-Tat-hMsrA is constructed; the recombinant plasmid is electrically transformed into a host, namely bacillus subtilis for strain culturing to obtain a positive expression fungus; and the positive expressionfungus is subjected to induction culture, expression product liquid containing Tat-hMsrA is obtained, digestion, renaturation and purification are conducted, thus the gene recombinant protein Tat-hMsrA is obtained. According to the production method of the gene recombinant protein Tat-hMsrA, the Tat-hMsrA with high activity can be obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for producing gene recombinant protein Tat-hMsrA. Background technique [0002] Methionine sulfoxide reductase A is another antioxidant enzyme that has attracted widespread attention after superoxide dismutase. Compared with superoxide dismutase, methionine sulfoxide reductase A not only has the function of removing oxidation factors in cells, but also can effectively repair damaged proteins. [0003] Methionine sulfoxide reductase A (MrsA) is a strong antioxidant enzyme. The cell-penetrating peptide Tat is safe and non-toxic, and can penetrate cells and the blood-brain barrier. The human-derived recombinant protein Tat-hMrsA obtained by combining the two can be expected to be used in cosmetics, health products and medicines. [0004] The paper "The Protective Effect of Methionine Sulfoxide Reductase A on Mitochondrial Oxidative Stress Damage and the Pilot-test Process of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N9/02C12R1/125
CPCC07K2319/10C12N9/0051C12N15/75C12Y108/04011
Inventor 曾建华闫云云胡雪平
Owner WUHAN HUA PEPTIDE BIOLOGICAL TECH CO LTD
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