Method for improving output of recombinant human Cu, Zn-SOD activated protein
A technology for active protein and yield, applied in the biological field, can solve the problem of low yield of active protein, and achieve the effect of simplifying the purification process, increasing the yield and increasing the yield.
Active Publication Date: 2009-09-09
陕西科聚美康生物科技有限公司
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And above-mentioned hCu, Zn-SOD is modified or select different expression vectors, although obtain the recombinant protein with activity, the output of the active protein finally obtained is not high
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[0050] The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.
[0051] In order to solve the problem of using genetic engineering to obtain biologically active rhCu, Zn-SOD protein, and improve the output of active protein, the scheme adopted by the present invention is to improve the ratio of soluble protein to total protein, and implement by the following scheme:
[0052] 1. Firstly, it is determined that increasing the ratio of rhCu, Zn-SOD in the supernatant of the lysate can greatly increase the activity yield of rhCu, Zn-SOD.
[0053] The hCu, Zn-SOD gene is cloned into the prokaryotic expression vector pET22b, and after induction, it can be effectively expressed in Escherichia coli, and the expression amount accounts for about 50% of the total amount of bacteria. After lysis with lysozyme, the proportion of the target protein in the supernatant of the lysozyme accounts for about 40% of the total amo...
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The invention relates to a method for improving the output of recombinant human Cu, Zn-SOD activated protein, which comprises the following steps: the site-directed mutagenesis to gene sequence for coding the human Cu, Zn-SOD protein is carried out; a human Cu, Zn-SOD recombinant expression carrier is built so as to convert host cells and improve the proportion of soluble recombinant protein expressed by the human Cu, Zn-SOD recombinant expression vector; and the site-directed mutagenesis adopts the way that the 6th cysteine residue and the 111th cysteine residue generates the site-directed mutagenesis to form hydrophilic amino acid residue. The 6th cysteine residue and the 111th cysteine residue are both mutated into hydrophilic serine to cause that the proportion of recombinant protein in schizomycete supernatant in the recombinant albumen total expression is improved to 80 percent from 40 percent, and the specific activity of mutant rhCu, Zn-SOD purified from the supernatant is equivalent to the specific activity of non-mutant rhCu, Zn-SOD purified in the supernatant.
Description
technical field [0001] The invention belongs to the field of biotechnology, in particular to gene site-directed mutagenesis, gene recombination, expression of foreign genes in prokaryotic cells and purification of target protein, in particular to a method for improving recombinant human Cu, Zn-SOD (rhCu, Zn-SOD) Methods for Active Protein Yield. Background technique [0002] 1. The discovery and mechanism of SOD [0003] Superoxide dismutase (Superoxide Dismutase, SOD) has the functions of anti-radiation, anti-tumor, and anti-oxidation, and is a medicinal enzyme with a wide range of curative effects. The enzyme was first isolated as a blue Cu-containing protein from bovine erythrocytes by Mann and Keilin in 1938, and it was only recognized as a protein at that time. Since Fridovich et al. confirmed in 1968 that it has the specific biological activity of scavenging oxygen free radicals in organisms, it has been widely valued. This enzyme widely exists in various tissues of...
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IPC IPC(8): C12N9/08C12P19/34C12N15/70C12N15/74C12R1/19
Inventor 万一张琨张月娟王琰
Owner 陕西科聚美康生物科技有限公司