Cloning And Expression Of Gene Encoding Transglutaminase, And Refolding Of The Enzyme

Microbial transgultaminase (protein-glutamine- γ-glutamyltransferase, EC2.3.2.13)is capable of catalyzing acyl transfer reactions which introduce covalent cross-linking between proteins and peptides by forming isopeptide bonds (ε- ( γ-glutaminyl) lysine bonds).It can improve the virtue of proteins.So it has potential application in food, cosmetic, pharmacy and other industries.The following aspects were studied in this thesis:1. With genomic DNA of streptoverticillium mobaraense as a template, a segment gene mtg encoding transglutaminase was obtained by PCR amplification and cloned into pQE-30T to construct a expression plasmid pMTG, then the plasmid pMTG was transformed into E.coli M15. The study of plasmid stability indicated the loss rate of plasmid was about 24 percent after subculture 5 times in the medium without Ampicillin, which showed that the pMTG was basically stable SDS-PAGE analysis revealed that with IPTG induction the strain bearing the plasmid expresses the target protein (38kD) and the molecular weight is same as the one expected, and expression protein is above 20% of the total cell protein.The purity of recombinant MTG were more than 80% by Ni-NTA column purification.2. The effect of different culture conditions on the production of rMTG and growth of E.coli Ml5 (pMTG) was studied. The optimal incubation conditions were as follows: No more than 50 mL operational volume in 250 mL flask; the cultivation temperature was 37C. the best induction was started when OD600 reached to 0.5 ~0.7 by adding 1 mmol/L of IPTG and the inducing time was 4 h.3. Inclusion bodies were formed after recombinant induction by IPTG. The purity of rMTG was 95% after purifying by washed and Ni-NTA column. The effect of Urea, glycine, GSH/GSSG and pH on refolding was studied for dilution refolding method. The optimal refolding conditions were as follows: glycine: 100 mmol/L, Urea: 1mol/L, GSH/GSSG: 10:1, pH9.5. Dialysis was carried out for refolding of rMTG by reducing concentration of Urea. The specific activity and total protein recovery of MTG were 13.63U/mg and 48.9%, respectively. The rMTG was refolded by gel filtration chromatography, The specific activity and the protein concentration of MTG were 21.3U/mg and 75mg/L.