紫红链霉菌2917磷脂酶A_2在大肠杆菌Rosetta(DE3)中表达与复性  

Expression and Renaturation of Streptomyces violaceoruber 2917 Phospholipase A_2 Protein in E.coil Rosetta(DE3)

作　　者：杨波[1] 邓贝[1] 刘志刚[1] 陈威[1] 李维琳[2] 胡征[1] 

机构地区：[1]湖北工业大学轻工学部,湖北武汉430068 [2]华中农业大学生命科学学院,湖北武汉430070

出　　处：《食品与发酵科技》2013年第2期5-8,共4页Food and Fermentation Science & Technology

基　　金：国家高技术研究发展计划(863计划)"食用油生物制造技术研究与开发"项目子课题"用于植物油脂精炼之磷脂酶的创制"项目编号(2010AA10501)

摘　　要：通过化学合成紫红链霉菌2917分泌型磷脂酶A2全基因序列后插入融合表达载体pGEX-6P-1中,经酶切和测序鉴定无误后转化E.coli Rosetta(DE3)菌株,用异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,获得目的蛋白后对包涵体形式的重组蛋白进行稀释复性,同时对复性后的蛋白进行酶学研究.结果表明磷脂酶A2基因在大肠杆菌中获得了高效表达(占总细胞蛋白30%),复性后酶液酶活性为0.15U/mL。这将为包涵体的复性研究以及磷脂酶A2结构和功能的研究打下良好的基础。The gene encoding the secreted phospholipase A2 of Streptomyces violaceoruber 2917 was chemical syn- thesized without changing the amino acid sequence and cloned into pGEX-6P-1 to construct the expression vector pGEX-6P-1-PLA2. Then, this recombinant plasmid was introduced into E. coli Rosetta （DE3） to obtain engineering bacterium. The fusion recombinant protein was expressed as inclusion body after induction of isopropy-β-D-thio- galactosic （IPTG）. The inclusion bodies were then harvested and refolded by means of pulsed-dilution method. Sub- sequently, the enzymatic activity of the refolded phospholipase A2 was determined. SDS-PAGE analysis showed that the phospholipase As was highly expressed in the E. coli cells, accounting for around 30% of total cell proteins. Enzymatic activity assay showed that this refolded protein displayed a activity of 0.15U/mL of enzyme. This study laid a foundation for the further studies of renaturation of the enzyme, and also its industrial application.

关 键 词：磷脂酶A2 复性 表达纯化 

分 类 号：Q814.9[生物学—生物工程] Q555.7