摘要
目的优化灰色链霉菌海藻糖合成酶(Trehalose synthase,TreS)基因密码子,提高海藻糖合成酶大肠杆菌基因工程菌株表达水平。方法基于大肠杆菌基因组密码子偏好数据表对tres基因进行密码子优化得基因tres1。基因工程法构建分别含tres及tres1基因的表达载体pET-26b(+)-tres和pET-26b(+)-tres1,CaCl2法转入大肠杆菌BL21(DE3)获得表达海藻糖合成酶的大肠杆菌基因工程菌株,采用异丙基硫代半乳糖苷(IPTG)诱导表达,对摇瓶发酵粗酶液进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析及酶活性测定以确定表达水平。结果 1707 bp海藻糖合成酶tres基因共优化碱基序列268 bp,优化后序列的GC含量由65.8%降低至54.4%,密码子适应指数(CAI)由0.37提升至0.97,SDS-PAGE及酶活性分析表明,含pet-26b(+)-tres1表达载体的大肠杆菌基因工程菌株海藻糖合成酶蛋白表达量高,酶活性比含pet-26b(+)-tres工程菌提高60.3%以上。结论密码子优化可有效提高海藻糖合成酶TreS蛋白在大肠杆菌中的表达,为海藻糖合成酶的异源高效表达提供了重要参考。
Objective To enhance the expression level of trehalose synthase(Tres) in Escherichia coli by optimization of gene codon. Methods Codon optimization of the tres gene was carried out based on the codon preference data of Escherichia coli genome. The tres and the optimized sequence tres1 gene were cloned to pET-26 b(+) respectively. The recombinant expression plasmid pET-26 b(+)-tres and pET-26 b(+)-tres1 were transformed into E. coli BL21(DE3).After inducation by IPTG, the expression levels of the two strains were analyzed by SDS-PAGE. The enzyme activity of crude enzyme solution was determined. Results 268 base pairs of tres gene(1707 base pairs) were optimized. The GC content of the new gene was reduced from 65.8 % to 54.4 %. The codon adaptation index(CAI) increased from0.37 to 0.97. SDS-PAGE and enzyme activity analysis showed that the expression of trehalose synthase protein in E.coli BL21(DE3) with plasmid pET-26 b(+)-tres1 was higher than that of the strain with plasmid pET-26 b(+)-tres, and the enzyme activity was increased by more than 60.3 %. Conclusion Codon optimization can effectively enhance the expression of trehalose synthase protein in E. coli. This method provides an important reference for heterologous expression of trehalose synthase.
引文
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