Studies On The Heterologous Expression Of MTG

Transglutaminases (protein-glutamine: amine y-glutamyltransferase, EC 2.3.2.13, TGase) widely distributed in most tissues, body fluids including the liver, hair follicles, epidermis, prostate, and blood. TGase is thought to be involved in diverse biological functions, such as maintenance of gross forms of structures and limited degrees of extensibility. TGase is also capable to enhance or change a wide variety of protein's binding properties, which has extensive potential applications in food industry and medical treatment.TGase gene from Streptoverticillium mobaraense (MTG )is cloned by PCR method. The sequence of MTG is the same as the literature. The expression vectors pET30a, pET22b, and pBV220 were used for the heterologous expression of MTG in E. colt. The highest expression level of MTG was obtained in the E.coli BL21 (DE3)/pET30a-MTG with a yield of 0.44 U/ml fermentation medium after 5 hours induction of ImM IPTG at 37℃. The recombinant MTG was confirmed by dot blotting and Western blotting. Investigation on the purification of MTG by Ni-affinity chromatography showed that 13mg purified MTG could be obtained from 1 liter fermentation medium with 69.2% recovery yield based on the the total protein. The specific activity of the purified MTG reached to 15.1U/mg MTG, which is the highest level among the reported genetic engineered strains. Experimental results on the stability and properties of MTG showed that under -80 癈 the optimized storage buffer, for the recombinant MTG is 25mM Tris-HCl (pH6.0) containing 0.5mM EDTA and lOmM DTT; the suitable pH and temperature were 6.5 and 40℃ respectively. The biological activity of recombinant MTG was verified by BSA conjunction reaction that was assayed by SDS-PAGE electrophoresis. The results showed that the recombinat MTG possessed the same activityas the native or commercialized MTG.