甘油脱氢酶基因在大肠杆菌中的密码子优化表达

Abstract

[中文文摘]【目的】利用密码子优化技术,提高甘油脱氢酶基因gldA在大肠杆菌中的表达水平。【方法】针对gldA起始密码子下游区域,优先选择AT含量最高的同义密码子,从而在不改变氨基酸序列的前提下,提高该区域的AT含量。利用大引物PCR的方法对野生型gldA-WT进行定点突变,获得优化型基因gldA-4,与pET-32a(+)连接后,构建表达质粒pET-gldA-4,转入E.coli BL21(DE3),得到工程菌E.coli-4。同时,设定包含gldA-WT的工程菌E.coli-WT作为对照,摇瓶发酵后,以甘油为底物检测比较表达产物的酶活力。【结果】gldA-4相对gldA-WT而言,改变了第2、5、6位密码子中的4个碱基,AT含量从53.3%提高到80.0%。相应地,E.coli-4的粗酶液的酶活力为191.3 U/mL,比E.coli-WT的48.3 U/mL提高了3倍多。【结论】本优化方案简便、快捷,但可明显提高甘油脱氢酶的酶活力,有望成为改善目的基因异源表达水平的一种通用方法。[英文文摘]Objective]To improve expression level of glycerol dehydrogenase gene gldA in Escherichia coli by means of codon optimization.[Methods]For immediately downstream region of initiation codon in gldA,we designed optimized sequence by choosing higher AT-content synonymous,in order that this region's AT-content was increased without changing the corresponding amino acids.Then we had wild gene gldA-WT site-directed mutagenesis depending on mega-primers PCR,so that physically optimized gene gldA-4 was acquired.We cloned gldA-4 into pET-32a(+) to construct expression plasmid pET-gldA-4，which was transformed into Escherichia coli BL21(DE3) for gaining engineering bacteria E. coli-4， by contrast engineering bacteria involved gldA-WT named E. coli-WT. After E. coli-4 and E. coli-WT were fermented in shake flasks，we measured enzyme activities of expression products with glycerol as substrate. ［Results］Four gldA-4's bases in the second，fifth and sixth codon were different with gldA-WT，so AT-content of the optimized gene was up to 80. 0% higher than the wild gene's 53.3% . Furthermore，enzyme activity of E. coli-4's crude extract was 191.3 U /mL more three times than E. coli-WT's 48.3 U /mL. ［Conclusion］This optimization scheme was quick and easy，but indeed increased dehydrogenase's activity. It possible becomes a universal method to improve heterogenous expression level of target genes.