Method for preparing acetylcoenzyme A synthetase in human pathogen clostridium difficile

A technology of Clostridium difficile and acetyl coenzyme, which is applied in the field of genetic engineering and protein expression, can solve the problems of no research reports and achieve the effect of increasing production and promoting assembly

Inactive Publication Date: 2013-04-10
FUDAN UNIV
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Problems solved by technology

Currently, there is no research report on ACS derived from Clostridium difficile

Method used

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  • Method for preparing acetylcoenzyme A synthetase in human pathogen clostridium difficile

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Embodiment Construction

[0021] The genomic DNA of Clostridium difficile 630, a human pathogen, was purchased from ATCC in the United States, and then the cDNA of ACS was obtained by genetic engineering, and it was successfully cloned into the pET30a(+) vector. The results of gene sequencing showed that we successfully constructed the expression plasmid of the pathogen Clostridium difficile 630ACS.

[0022] Transformed with BL21(DE3) as the host bacteria, picked spots and cultured in LB medium overnight, and then transferred the bacterial solution to 8L LB medium according to the ratio of 1:100 (add 4g / L glucose, 100μg / mL kanamycin prime), 37°C, 250rpm to OD 600 ~0.7, then fill with nitrogen, add 0.1mM ferric ammonium sulfate, 2mM cysteine, 1.5mM sodium sulfide after 30 minutes, and continue to cultivate. When OD 600 ~0.8, add IPTG (final concentration 0.1mM) and 0.2mM nickel dichloride, and at the same time reduce the temperature to 20°C, express for ~12h, and harvest the bacteria. Resuspend the b...

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Abstract

The invention belongs to the technical field of bioengineering, and specifically provides a method for preparing acetylcoenzyme A synthetase (ACS) in human pathogen clostridium difficile. The invention utilizes gene engineering technology to successfully get expression plasmid of acetylcoenzyme A synthetase target gene in human pathogen clostridium difficile 630, shifts the plasmid into escherichia coli host bacteria for culture and fermentation, and establishes an efficient expression-purification method for acetylcoenzyme A synthetase target gene in human pathogen clostridium difficile 630 for the first time. The method can obtain clostridium difficile 630 acetylcoenzyme A synthetase protein of which the purity is up to over 90 percent and the yield is up to over 10 mg / L, and is the only expression method reported at present. The inhibitor is of important application value in the field of biological medicine.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and protein expression, and in particular relates to a method for efficiently preparing acetyl-CoA synthetase (ACS) in human pathogen Clostridium difficile (clostridium difficile) by bioengineering. Background technique [0002] Acetyl coenzyme A is an important substance in the metabolism of matter and energy, and it is a pivotal substance in the metabolism of energy substances in the body. Acetyl-CoA is one of the important intermediate metabolites in the tricarboxylic acid cycle, a metabolic pathway commonly found in aerobic organisms; while in some anaerobic microorganisms such as acetogenic bacteria and methanogenic bacteria, it mainly passes through the Wood-Ljungdahl pathway, Rely on CO / CO 2 and H 2 As a carbon source and energy source, it synthesizes acetyl coenzyme A for material and energy metabolism. [0003] Acetyl-CoA synthetase (ACS) catalyzes the synthesis of acetyl-C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N9/00C12R1/145C12R1/19
Inventor 谭相石朱小飞
Owner FUDAN UNIV
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