Method for improving extracellular secretion level of recombinant protein of Escherichia coli

A technology of Escherichia coli and recombinant protein, applied in the field of genetic engineering and fermentation engineering, can solve the problems of hindering protein production and accumulating a large amount of intermediates

Inactive Publication Date: 2019-01-18
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[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 pro

Method used

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  • Method for improving extracellular secretion level of recombinant protein of Escherichia coli
  • Method for improving extracellular secretion level of recombinant protein of Escherichia coli
  • Method for improving extracellular secretion level of recombinant protein of Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 This example illustrates the construction process of dacA and dacB knockout Escherichia coli

[0027] (1) Knock out dacA and dacB respectively:

[0028] Using the plasmid pKD13 as a template, design primers and PCR amplify the homologous fragments containing the Kan resistance gene used to replace the target genes dacA and dacB. The sequences are shown in SE ID NO.1 and SEQ ID NO.2, respectively. Electroporation In E.coliBL21(DE3) / pKD46 competent state, spread on a plate containing kanamycin (Kan) to obtain transformants BL21-ΔdacA::kan and BL21-ΔdacB::kan. Colony PCR amplification was used to verify whether the dac gene was successfully knocked out. In theory, BL21-ΔdacA::kan and BL21-ΔdacB::kan should contain homologous fragments of 1504 and 1754bp, respectively, such as figure 1 As shown, the fragment size obtained by colony PCR is consistent with the theoretical homologous fragment size. The pCP20 helper plasmid was used to eliminate the kan gene. After ...

Embodiment 2

[0031] Example 2 This example illustrates the effect of knocking out dacA and dacB on the extracellular production of recombinant green fluorescent protein in E.coli

[0032] In this example, the effect of gene knockout on the extracellular production of GFP in E. coli was determined and analyzed. The recombinant plasmid pETDuet-gfp was respectively transformed into the BL21-ΔdacA, BL21-ΔdacB and BL21-ΔdacA / B strains constructed in Example 1 to construct recombinant mutants BL21-ΔdacA-gfp, BL21-ΔdacB-gfp and BL21-ΔdacA / B B-gfp. Recombinant mutants BL21-ΔdacA-gfp, BL21-ΔdacB-gfp and BL21-ΔdacA / B-gfp were cultured in LB medium at 37°C for 8 hours, 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. Measure the amount of GFP fluorescence. Such as figure 2 As shown, the extracellular GFP fluorescence values ​​of BL21-ΔdacA-gfp, BL21-...

Embodiment 3

[0033] Example 3 This example illustrates the effect of knocking out dacA and dacB on the extracellular production of recombinant amylase in E.coli

[0034] In this example, the recombinant plasmid pETDuet-amyk was transformed into BL21-ΔdacA, BL21-ΔdacB and BL21-ΔdacA / B mutant strains respectively to obtain recombinant mutant strains BL21-ΔdacA-amyk, BL21-ΔdacB-amyk and BL21-ΔdacA / B B—amyk. BL21-ΔdacA-amyk, BL21-ΔdacB-amyk and BL21-ΔdacA / B-amyk were cultured in LB medium at 37°C for 8 h, and then inoculated into TB medium at a 1% (v / v) inoculation amount, Cultured at 37℃ to OD 600 =0.8, the final concentration of adding is 1mmol·L -1 IPTG, induced expression at 25°C. After the fermentation, centrifuge and collect the fermentation supernatant, detect the amylase activity in the fermentation supernatant, and analyze the effect of knocking out dacA and dacB genes in E.coli on the production of extracellular recombinant amylase.

[0035] Such as image 3 As shown in (a), aft...

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Abstract

The invention discloses a method for improving the extracellular secretion level of recombinant protein of Escherichia coli, belonging to the field of genetic engineering and fermentation engineering.The invention knocks out D. Alanyl-D-Alanine carboxypeptidase dacA and dacB genes (single knockout and double knockout) were cloned into recombinant Escherichia coli mutants with significantly increased extracellular secretion of recombinant proteins. Green fluorescent protein (GFP) and amylase are adopt as model recombinant proteins, which prove that that method can remarkably increase the content of extracellular proteins. The extracellular fluorescence of GFP was increased by 2.1, 2.1 and 2.7 times after single knockout and combined knockout of dacA and dacB, respectively. After single knockout and combined knockout of dacA and dacB, the proportion of extracellular amylase activity to total enzyme activity increased from 40.7% to 44.7%, 62.1% and 64.2%, respectively. The method of theinvention has important guiding significance for high-efficient secretion and production of the recombinant protein in Escherichia coli.

Description

technical field [0001] The invention relates to a method for increasing the extracellular secretion level of Escherichia coli recombinant protein, which belongs to the field of genetic engineering and fermentation engineering. 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 β-1,4 glycosidic bonds, and the s...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N9/26C12R1/19
CPCC12N9/2411C12N9/485C12N15/70C12Y304/17014
Inventor 陈献忠杨海泉沈微卢潇胡金远王拂祥王浩坤陈媛
Owner JIANGNAN UNIV
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