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Plasmid vector containing ethanol induced promoter and application thereof in increasing expression level of recombinant protein of corynebacterium glutamicum

A Corynebacterium glutamicum, plasmid vector technology, applied in the field of genetic engineering and metabolic engineering, to achieve the effect of improving expression and self-induction effect

Pending Publication Date: 2018-10-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the expression system of Corynebacterium glutamicum, people are still limited to the use of traditional promoters such as tac, trc, and cspB. The induction fold is only 3.5 times, which shows that the application of Corynebacterium glutamicum self-inducible promoter in protein expression has yet to be developed

Method used

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  • Plasmid vector containing ethanol induced promoter and application thereof in increasing expression level of recombinant protein of corynebacterium glutamicum
  • Plasmid vector containing ethanol induced promoter and application thereof in increasing expression level of recombinant protein of corynebacterium glutamicum
  • Plasmid vector containing ethanol induced promoter and application thereof in increasing expression level of recombinant protein of corynebacterium glutamicum

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Experimental program
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Effect test

Embodiment 1

[0025] Effect of ethanol addition on protein expression of Corynebacterium glutamicum:

[0026] The Corynebacterium glutamicum strain expressing EGFP (pXMJ19-EGFP) was taken out from the -80°C refrigerator, and after being streaked on a solid plate for activation, the bacteria were picked and inoculated into 10 mL of LBB medium, 200 r / min, 30°C, and cultured for 24 hours. Transfer the above-mentioned bacterial solution to 2 bottles of LB liquid medium containing 10 mL with a 2% inoculation amount, add absolute ethanol with a final concentration of 0.5% to 3% to one of the above two culture bottles, and the other The bottle is not added. 200r / min, 30°C, cultured for 36h. The above-mentioned bacteria were collected, and the amount of the bacteria was determined to be consistent. The protein was extracted by ultrasonic crushing, and the above-mentioned protein samples were subjected to SDS-PAGE electrophoresis. It was found that the addition of 1% ethanol would make the intrace...

Embodiment 2

[0031] Construction of ethanol-induced promoter expression vector and its effect on the expression of foreign protein EGFP:

[0032] Primer P ICL -B-EGFP-UF / R performs PCR amplification on the Corynebacterium glutamicum genome to obtain the promoter region of the isocitrate lyase encoding gene with HindⅢ / and bsaⅠ linker (from ATG upstream 500bp to ATG downstream 59bp , including the transcription initiation site and the complete 5' untranslated region). Primer P ICL -B-EGFP-DF / R The EGFP-containing plasmid pXMJ19-EGFP was amplified by PCR to obtain an EGFP fragment containing bsaI and BamHI linkers (its 5' end contains a conserved ribosome binding site AAAGGAGGA). The obtained promoter fragment and EGFP fragment were respectively treated with the endonucleases corresponding to the above linkers. After digestion and purification, the two fragments were ligated with the p19-0 vector treated with HindⅢ and BamHI. Transform Escherichia coli DH5α to construct the ethanol-induc...

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Abstract

The invention discloses a plasmid vector containing an ethanol induced promoter and application thereof in increasing the expression level of a recombinant protein of corynebacterium glutamicum. The ethanol induced promoter is the promoter of the coding gene of isocitrate lyase of the corynebacterium glutamicum. A vector loaded recombinant protein of the ethanol induced promoter is contained, after the plasmid vector is cultured for 36 hours in an LBB culture medium containing 1% of ethanol, the plasmid vector has a 9.5-time stronger induction effect compared with the ethanol induced promoterof the plasmid vector to which ethanol is added. The plasmid vector containing the ethanol induced promoter has a significant effect on improving the expression of the recombinant protein, and the ethanol induced promoter has an improved 21.1-time stronger self-induction effect compared with a self logarithmic phase expression level (4h).

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and metabolic engineering, and in particular relates to a plasmid vector containing an ethanol-induced promoter and its application in improving the expression of Corynebacterium glutamicum recombinant protein. Background technique [0002] Corynebacterium glutamicum is a facultative anaerobic Gram-positive industrial microorganism widely used in the synthesis of some organic acids. Due to its advantages of no endotoxin, strong stress resistance, and low protease level, Corynebacterium glutamicum is gradually being developed for the production of biofuels and the expression of exogenous recombinant proteins in recent years. [0003] A promoter is a DNA sequence that is specifically recognized and bound by RNA polymerase. It is an integral part of a gene and controls the initiation of gene transcription. The strength of the promoter will directly affect the protein expression level. S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N15/67
CPCC12N15/67C12N15/77C12N2830/002
Inventor 刘秀霞高雄孙曼曼张伟董贵宾杨艳坤白仲虎
Owner JIANGNAN UNIV
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