Recombinant Prokaryotic Expression Of Glutamine

Transglutaminases (TGase; protein-transglutayltransferase, EC 2.3.2.13), a new type enzyme preparation, are widely used in food and medical industries. This subject is paid attention to by the domestic and foreign scholars to study on it. In 1993, Japan turned TG into a kind of commodity, and got the huge economic benefits. But at the same time, the research and development of TG was still in the beginning stage in china. A strain with high productivity of MTG was obtained from soil by our research group. It was identified as Streptomyces griseoverticillatus, and named as Streptomyces H197. The maximum of MTG activity in the culture broth was 1.74U/mL.In order to promote a industrial development and TG`s application exploitation, using genetic engineering, The MTG gene from Streptomyces sp H197 was cloned to pET-30a and then to E.coli BL21 (DE3) and got the recombinant strain E.coli BL21/ pET-30a-MTG (DE3).The recombinant strain expressed MTG in the form of inclusion body included by IPTG. According to the experiment, it showed that the optional condition for the expression of recombined MTG Gene′s were that the final concentration were 1.0 mmol/L IPTG, the temperature were 37℃, the shaking condition were 250 r/min for 4 h. Under the Optimized conditions, the target protein expression increased 10%.After extracted crude inclusion body proteins, such as Triton X-100 and lysozyme were added for cleaning crude enzyme lysozyme, and they helped to remove other hybrid protein. The recombinant MTG formed of fusion protein, and it contained His-tags, so affinity chromatography was used to purify and improved the purity of the target protein, while removed the non-fusion protein fragments. By the dialysis and the column chromatography, the biological activities of the recombinant MTG were recovered, and the enzyme activities were 0.42U/ml and 0.71U/ml, and the specific activities were 0.182U/mg and 0.236U/mg. The results showed that the column was better than the dialysis for refolding efficiency.In order to achieve the industrial production, this paper groped some strategies for recombinant MTG`s soluble expression. Firstly, it could use other inducers instead of IPTG. The lactose which is the analog of IPTG is cheaper than IPTG, and with the advantage of no contaminates and low cost. The results indicated that the recombinant protein was mostly soluble, but the expression decreased significantly. Secondly, it induced at lower temperature. The equal samples were induced at different temperatures. The results indicated that the expression(2.0801mg/ml)of the target protein in the soluble form reached the maximum at 30℃, but the expression decreased significantly, compared to the expression in the form of inclusion body.All of the work not only provides the theory basis and the technical references to mass-producing recombinant MTG, but also establishes the laboratory basis of industrialized production of MTG.