转谷氨酰胺酶酶原在大肠杆菌中的重组优化表达

茂原链霉菌(Streptomyces mobaraensis)转谷氨酰胺酶酶原(pro-transglutaminase,pro-TG)在重组大肠杆菌中的表达量低,限制了其在食品、化妆品、纺织行业中的应用。通过对转谷氨酰胺酶酶原的基因序列进行密码子优化,降低其GC含量,提高了它在大肠杆菌中的表达水平,结果表明经优化后转谷氨酰胺酶原的表达量达到了优化前的4.4倍。为提高转谷氨酰胺酶的比活力,通过基于融合PCR的定点突变技术将转谷氨酰胺酶第二位的丝氨酸突变为脯氨酸,突变后转谷氨酰胺酶的比活力达到了突变前的1.26倍。上述研究结果表明,密码子优化及定点突变技术可以应用于优化转谷氨酰胺酶酶原在大肠杆菌中的表达。

Recombinant expression and optimization of pro-transglutaminase gene in Escherichia coli

The pro-transglutaminase gene was subjected to codon usage optimization and then expressed in E.coli.Results demonstrated that GC content of the optimized gene decreased and its expression level was improved by 3.4-fold than the unoptimized gene.In order to improve the specific activity of transglutaminase,the serine at the second site of transglutaminase was mutated to proline by site-directed mutagenesis technology.The specific activity was 1.26-fold of the unmutated gene.The above results suggested that codon usage optimization and site-directed mutagenesis technology could be utilized to improve the expression level of pro-transglutaminase in E.coli.