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Efficient culture medium for expressing soluble recombinant protein by escherichia coli and preparation method of culture medium

A technology of recombinant protein and Escherichia coli, which is applied in the biological field, can solve the problems of complex and difficult renaturation process, easy to produce glucose effect, and reduced utilization rate of lactose

Inactive Publication Date: 2020-05-08
邵国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of medium is rich in nutrients, and the bacteria can grow normally and rapidly. It is used in various cultures, but in the process of prokaryotic induced expression, it is easy to produce glucose effect.
That is, too much glucose will inhibit the growth and production of microorganisms, reduce the utilization rate of lactose, and cannot normally open the transcription of genes, which will affect the subsequent translation and synthesis of proteins
Especially under the optimal temperature conditions for bacterial growth, exogenous proteins in LB medium are easily expressed in the prokaryotic cytoplasm to form inclusion bodies and the renaturation process is complicated and difficult. Misfolding or failure to form disulfide bonds will lead to changes in protein structure. affect the biological activity of the protein

Method used

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  • Efficient culture medium for expressing soluble recombinant protein by escherichia coli and preparation method of culture medium
  • Efficient culture medium for expressing soluble recombinant protein by escherichia coli and preparation method of culture medium
  • Efficient culture medium for expressing soluble recombinant protein by escherichia coli and preparation method of culture medium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] 1. The operation process of recombinant protein expression

[0112] a) Pick a single clone and place it in 2ml LB overnight (pGEX-4T plasmid is added with 0.1% ampicillin, and other plasmid antibiotics are selected based on the resistance of the plasmid, such as pET-21, with 0.1% kanamycin);

[0113] b) Transfer to 50ml M9+LB (4:1), add corresponding antibiotics, add IPTG to 1mM, shake at 4°C until turbid;

[0114] c) Centrifuge, remove the supernatant, wash the pellet with PBS, centrifuge, and remove the supernatant;

[0115] d) Suspend in PBS, sonicate (about 100 times), and pass through the column (depending on the label, choose different columns, such as pGEX-4T, use GST columns; and pET-21 use nickel columns). Wash the column with 5-10 times volume of PBS;

[0116] e) Use the eluent to elute the target protein, add PMSF and store at 4°C. The composition of the eluent depends on different expression vectors. For example, pET-21 vector is eluted with imidazole-containing elue...

experiment example

[0122] Transform the human DNMT3B gene into the pGEX-4T prokaryotic expression vector, and then transfer the recombinant plasmid into competent E. coli, spread it on a plate containing amp and incubate at 37°C for 16 hours, pick a single clone and inoculate it into 2ml of liquid LB containing amp In the medium, cultured at 225 rpm 37°C for 16 hours, then transferred to 200 ml of the high-efficiency medium of the present invention, cultured at 225 rpm 4°C for 72 hours. Centrifuge at 1000 RPM for 10 minutes, discard the supernatant, wash the pellet with 6 times volume of PBS 3 times, then add 1 volume of PMSF-containing PBS, and ultrasonically break. Centrifuge at 12000rpm for 10 minutes, add an equal volume of 80% glycerol to the supernatant and store at -80°C.

[0123] Such as figure 1 As shown, the foreign protein is DNMT3B, approximately 120KDa. According to the staining results, the expression level of DNMT3B in the ordinary medium is very low, while the expression level of ...

Embodiment 2

[0125] A high-efficiency culture medium for expressing soluble recombinant protein in Escherichia coli, which is characterized in:

[0126] The high-efficiency medium selected M9 medium, added sorbitol, ethanol, glucose, sucrose [etc.] basic ingredients, supplemented with magnesium sulfate and ampicillin, IPTG as an inducer, aimed at increasing the soluble expression of foreign proteins .

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Abstract

The invention belongs to the technical fields of genetic engineering, molecular biology and other biology, and particularly relates to an efficient culture medium for expressing a soluble recombinantprotein by escherichia coli and a preparation method of the culture medium. The culture medium can improve the success rate and yield of expressing the soluble recombinant protein in cytoplasm by theescherichia coli; the efficient culture medium is selected from an M9 culture medium, basic ingredients such as sorbitol, ethanol, glucose and sucrose are added, magnesium sulfate and ampicillin are also added, IPTG is used as an inducer, and the efficient culture medium aims to increase the soluble expression of the foreign protein; the efficient culture medium is used to express the recombinantprotein, especially to express the soluble recombinant protein by the escherichia coli; and the culture medium can use escherichia coli BL-21 or DH5alpha strains as reaction bacteria, or K12 or origamiB escherichia coli strains as reaction bacteria.

Description

Technical field [0001] The invention belongs to the fields of biological technology such as genetic engineering and molecular biology, and specifically relates to a high-efficiency culture medium for expressing soluble recombinant protein in Escherichia coli and a preparation method thereof. The medium can improve the success rate of Escherichia coli expressing soluble recombinant protein in the cytoplasm. And yield. Background technique [0002] Escherichia coli belongs to the kingdom of bacteria, Proteobacteria, Enterobacteriaceae, Escherichia, and Escherichia coli. It is a typical Gram-negative bacteria. Because of its wide distribution in nature, simple structure, easy cultivation, fast reproduction speed, and clear genetic background, it has become one of the model organisms in biological research and is widely used in gene function research and gene expression product acquisition. It is widely used in the fields of genetics, molecular biology and genetic engineering. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/38C12P21/00C12R1/19
CPCC12N1/20C12N1/38C12P21/00
Inventor 邵国巴德仁贵鲍牧兰姜树原贾小娥谢伟谢雅彬刘晓蕾许文强石蕊
Owner 邵国
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