番茄Metacaspase基因(LeM)的克隆及其在大肠杆菌Rosetta中的高效表达  

Cloning of Lycopersicon esculentum Metacaspase Gene(LeM) and Its Effective Expression in Escherichia coli Rosetta

作　　者：马秋敏[1] 张雅丽[1] 刘金娜[1] 车英慧[1] 谢寒丁 罗云波[1] 曲桂芹[1] 

机构地区：[1]中国农业大学食品科学与营养工程学院食品生物技术实验室,北京100083

出　　处：《农业生物技术学报》2011年第2期258-262,共5页Journal of Agricultural Biotechnology

基　　金：中央高校基本科研业务费专项资金(No.2009-1-83);国家公益性行业(农业)科研专项(No.200803033-B0602-03);国家自然科学基金(No.30500352)共同资助

摘　　要：为获得可溶性的番茄Metacaspase蛋白,后续研究Metacaspase蛋白的酶学特性以及功能,利用RT-PCR方法克隆了番茄(Lycopersicon esculentum)中Metacaspase(LeM)全长cDNA,并成功地构建了LeM基因的原核表达载体重组质粒pET28a-LeM,转入大肠杆菌(Escherichia coli)BL21(DE3)PlysS和Rosetta中进行诱导表达,并对诱导表达条件进行了优化。SDS-PAGE结果显示,在37℃条件下,1mmol/L IPTG诱导表达量最高,在BL21(DE3)PlysS菌株中重组蛋白主要以包涵体形式存在,而相同条件下在Rosetta菌株中多为可溶性表达。同时酶活测定显示,Rosetta上清中LeM的活性为33RFU/min,显著高于BL21(DE3)PlysS 7RFU/min。本实验表明,不同菌株对Metacaspase的可溶性表达有很大影响。To obtain the fusion protein of Lycopersicon esculentum Metacaspase（LeM） and study the characteristics and function of it,we cloned the open reading frame of LeM in Lycopersicon esculentum by RT-PCR,and successfully constructed the prokaryotic expression vector pET28a-LeM for LeM expression.The recombinant plasmid pET28a-LeM was transformed into Escherichia coli BL21（DE3）PlysS and Rosetta for expression,induced by IPTG,and the conditions of the expression was optimized.SDS-PAGE revealed that the best expression was induced by 1 mmol/L IPTG at 37℃ and the recombinant protein expressed mainly as inclusion body in E.coli BL21（DE3）PlysS while as fusion protein in E.coli Rosetta at the same condition.Enzyme activity assay showed that the activity of LeM in E.coli Rosetta was 33 RFU/min,which was much higher than 7 RFU/min of E.coli BL21（DE3）PlysS.We demonstrate that the strain has a great influence on the expression of LeM fusion protein.

关 键 词：番茄 METACASPASE 基因克隆 原核表达 

分 类 号：S641.2[农业科学—蔬菜学]