Colibacillus and method for performing soluble expression of transglutaminase proenzyme thereof

Abstract

The invention discloses colibacillus and a method for performing soluble expression of transglutaminase proenzyme thereof. The method comprises the following steps: (1) designing PCR primer to perform PCR amplification according to the nucleotide sequence of transglutaminase proenzyme of streptomyces mobaraensis, cloning the related gene to prokaryotic expression vector pET22b (+),constructing expression vector pET22b-proMTG to conform to E.coli BL21 / proMTG of which strain preservation number is CCTCC M 208240; (2) performing soluble expression of transglutaminase proenzyme; (3) activating transglutaminase proenzyme; (4) performing high density fermentation; and (5) performing protein purification technology. In the method of the invention, the yield and specific activity of transglutaminase proenzyme can reach the level of that of transglutaminase proenzyme which is produced by firstly performing direct expression in colibacillus to form inclusion body and then performing denaturation and renaturation and the fermentation process is easier.

Description

technical field [0001] The present invention relates to transglutaminase genetic engineering, in particular to a method for soluble expression of transglutaminase zymogen in Escherichia coli to realize soluble expression of transglutaminase zymogen for large-scale industrialized production of transglutaminase enzyme. Background technique [0002] Transglutaminase is a transferase that catalyzes the acyl transfer reaction. It mainly catalyzes the amido transfer reaction between the γ-amide group of glutamine residue and the ε-amino group of lysine in proteins and polypeptides. Carry out covalent cross-linking polymerization between molecules, thereby changing the structure and function of the protein itself and the attached cells and tissues. The enzyme has been widely used in the pharmaceutical industry, food industry and textile industry. It has the reputation of "21st century super adhesive". advantage, which has attracted people's attention. In Japan, the food sales re...

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Problems solved by technology

One is to increase the yield of production bacteria through mutation breeding and medium optimization. For example, Japan Ajinomoto Company uses Streptomyces Mobara to ferment and produce MTG. However, the high-yielding bacteria obtained by this method are easy to degrade and the production is unstable; the second is By constructing genetically engineered bacteria to express transglutaminase, as published in "Journal of Biological Engineering" in the fifth issue of volume 21 in September 2005, "Streptomyces mobara transglutaminase gene is highly expressed in Escherichia coli" ( Authors: Xu Bin, Han Zhibo, Yang Ping, Liu Yongjun, Li Yanhan, Han Zhongchao) constructed a genetically engineered bacterium that successfully expressed transglutaminase, and reported that a microbial transglutaminase was amplified from the genomic DNA of Streptomyces mobara by PCR method. Gene fragment of glutaminase, instead of transglutaminase progene, and construct expression plasmid pET-MTG

The latter is highly expressed in Escherichia coli (Rosetta DE3), but the expressed MTG exists in the inclusion body, which needs to be washed, denatured and refolded, and purified by strong cation exchange chromatography to obtain SDS-PAGE Pure MTG, so the process is very cumbersome and difficult to achieve industrial production

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