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Method for increasing the extracellular secretion level of Escherichia coli recombinant protein

A technology of Escherichia coli and recombinant protein, which is applied in the field of genetic engineering and fermentation engineering, can solve the problems such as the unclear effect of recombinant protein extracellular secretion, and achieve the effect of changing permeability and increasing secretion level

Active Publication Date: 2022-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, how to set the position of the SD sequence and the distance between it and the start codon AUG in different strains, so the effect on the extracellular secretion of the recombinant protein is not very clear

Method used

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  • Method for increasing the extracellular secretion level of Escherichia coli recombinant protein
  • Method for increasing the extracellular secretion level of Escherichia coli recombinant protein
  • Method for increasing the extracellular secretion level of Escherichia coli recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction process of recombinant Escherichia coli integrating different numbers of SD sequences

[0058] (1) Using the plasmid pKD13 as a template, design primers and PCR amplify the homologous fragments containing the Kan resistance gene for replacing the 1SD, 2SD, 3SD, and 4SD sequences. The sequences are respectively as SEQ ID NO.1, SEQ ID NO. 2. As shown in SEQ ID NO.3 and SEQ ID NO.4, the Kan resistance gene contains FRT sites on both sides, and different SD sequence integration frame fragments are obtained.

[0059] (2) Transform the pKD46 plasmid into E.coli BL21(DE3) competent cells to obtain E.coli BL21(DE3) / pKD46 strains, prepare electroporation competent cells, and electroporate the integrated frame fragments prepared in step (1) Transformed into E.coli BL21(DE3) / pKD46 competent medium, the transformation solution was post-cultivated and spread on LB solid medium containing kanamycin (Kan) and ampicillin to obtain the transformant BL21::1SD: :ka...

Embodiment 2

[0061] Example 2 The effect of different distances from gene integration SD sequence to the initial codon ATG of carboxypeptidase DacA on the extracellular production of recombinant protein in E.coli

[0062] In this example, the effect of different distances from the gene integration 1SD sequence to the initial codon ATG of carboxypeptidase DacA on the extracellular production of recombinant protein by E. coli was determined and analyzed. In this example, GFP was used as a model protein for verification. Recombinant strains with distances from the initial codon ATG of DacA of 0, 2, 4, 6, 8, and 10 bases were respectively constructed to obtain recombinant strains BL21(+1SD), BL21(+1SD)-2, BL21(+1SD )-4, BL21(+1SD)-6, BL21(+1SD)-8, BL21(+1SD)-10, and transform the recombinant plasmid pET28a-gfp respectively. Such as figure 2 As shown, the extracellular secretion level of BL21(+1SD)-gfp recombinant GFP is the highest when the distance is 0 bases, and its extracellular GFP flu...

Embodiment 3

[0063] Example 3 Extracellular Production of Recombinant Green Fluorescent Protein by Recombinant Escherichia coli

[0064] In this example, the effect of gene integration on the extracellular production of GFP in E. coli was determined and analyzed.

[0065]The recombinant plasmid pET28a-gfp was respectively transformed into the starting strain BL21, the BL21(+1SD) and BL21(+2SD) strains constructed in Example 1, and the recombinant mutants BL21-gfp, BL21(+1SD)-gfp and BL21( +2SD)-gfp. The recombinant mutants BL21-gfp, BL21(+1SD)-gfp and BL21(+2SD)-gfp were cultured in LB medium at 37°C for 8h, and then inoculated into TB medium at an inoculation amount of 1% (v / v) medium, cultured at 37°C to OD 600 =0.8, the final concentration of adding is 1mmol·L -1 IPTG, induced expression at 25°C.

[0066] Measure the amount of GFP fluorescence. Such as image 3 As shown, the extracellular GFP fluorescence values ​​of BL21(+1SD)-gfp and BL21(+2SD)-gfp mutant strains were 2.8×10 5 ...

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Abstract

The present invention relates to a method for improving the extracellular secretion level of Escherichia coli recombinant protein, which integrates a gene fragment comprising 1-4 SD sequences into the encoding D-alanyl-D-alanine carboxypeptidase by means of homologous recombination Between the promoter of DacA and the gene encoding DacA. The present invention improves the expression of D,D-carboxypeptidase DacA by integrating different SD sequences between the promoter of the Escherichia coli DacA gene and the DacA coding gene, thereby increasing the extracellular secretion level of the Escherichia coli recombinant protein.

Description

technical field [0001] The invention relates to the fields of genetic engineering and fermentation engineering, in particular to a method for increasing the extracellular secretion level of Escherichia coli recombinant protein and recombinant Escherichia coli. Background technique [0002] The Escherichia coli expression system is currently the most commonly used recombinant protein expression system in genetic engineering. It has the advantages of simple operation and low cost, and can quickly and large-scale produce the target protein. However, in the heterologous expression of E. Signal peptide-guided secretion into the periplasmic space will lead to a large accumulation of intermediates, hindering protein production. [0003] Peptidoglycan is a multi-layer network macromolecular structure composed of disaccharide units, tetrapeptide tails and peptide bridges. The backbone of the peptidoglycan layer is composed of N-acetylglucosamine and N-acetylmuramic acid connected by...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/57C12N1/21C12N9/04C12N9/08C12N9/10C12N9/16C12N9/26C12N9/48C07K14/78C07K14/435C12P21/02C12R1/19
CPCC12N15/70C12N9/485C12N9/0006C12N9/0065C12N9/1051C12N9/2411C12Y204/01243C12Y111/01006C12Y101/03004C07K14/43595C07K14/78C12N9/16C12Y301/08001C12P21/02
Inventor 陈献忠杨海泉王浩坤张坤杰王拂祥沈微夏媛媛
Owner JIANGNAN UNIV
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