粘质沙雷氏菌PL-06磷脂酶A1基因大肠杆菌优化表达  

The Optimized Phospholipase A1 Gene Expression of Serratia marcescens PL-06 in E.coli

作　　者：苏燕南[1] 薛正莲[1] 陈涛[1] 马琦亚[1] 

机构地区：[1]安徽工程大学生物与化学工程学院微生物发酵安徽省工程技术研究中心,芜湖241000

出　　处：《中国生物工程杂志》2013年第7期36-42,共7页China Biotechnology

基　　金：安徽省自然科学基金(11040606M81);安徽省高校自然科学基金(KJ2009A168)资助项目

摘　　要：以粘质沙雷氏菌PL-06基因组为模板扩增得到磷脂酶A1基因plaA和含有辅助蛋白的磷脂酶A1基因plaB。plaA和plaB基因与pET-28a(+)连接后转入大肠杆菌BL21(DE3)表达,得到基因工程菌AP28和BP28。AP28最佳的诱导表达条件为诱导初始OD600值0.5,IPTG浓度为0.2mmol/L,诱导温度为37℃,诱导时间为4h,优化后磷脂酶A1蛋白表达水平从32%上升至46%,包涵体复性的磷脂酶A1酶活从10.8U/ml上升到12U/ml,相对于BP28工程菌,目的蛋白的表达对宿主细胞毒性小,而且得到的蛋白容易纯化。因此通过优化磷脂酶A1基因plaA的诱导条件,使磷脂酶A1以大量的包涵体形式表达,从而得到较高活性的磷脂酶A1并避免其对宿主细胞的毒性是可行的,而且可以得到大量纯化的磷脂酶A1蛋白,方便下一步的研究。The phopholipase A1 gene from Serratia marcescens PL-06 was named plaA,and the phospholipase A1 with accessory protein was plaB.The genes plaA and plaB were cloned,ligased with pET-28 a(+)and transferred to BL21(DE3).The engineered stains expressing plaA and plaB was obtained,and named AP28 and BP28.The phospholioase A1 expression level reached peak when the IPTG concentration was 0.2mmol/L,the OD600 was 0.5,the inducing temperature was 37℃ and the inducing time was 4 hours.Relatively the PLA1 protein expression level of AP28 rose from 32% to 46%,and the enzyme activity of refolded inclusion body enhanced from 10.8 U/ml to 12 U/ml.Contrast to BP28,the expression of target protein was low toxic to the host bacterial and easy to purify.Therefore,it is feasible that phospholipase A1 gene plaA expressed in the form of a large number of inclusion bodies by optimizing the inducing conditions,and thereby obtain higher activity of phospholipase A1 and avoid toxicity to the host cell,and it could obtain large amount of phospholipase A1 protein for the subsequent studies.

关 键 词：粘质沙雷氏菌 磷脂酶A1 诱导表达 包涵体复性 细胞毒性 

分 类 号：Q78[生物学—分子生物学]