大肠杆菌丙酮酸脱氢酶的克隆与表达优化  

Cloning and Optimization Expression of E.coli Pyruvate Dehydrogenase

作　　者：李艳[1] 谭宏亮[2] 何亚辉[1] 袁均林[1] 贺红武[3] 杨旭[1] 丁书茂[1] 李中振[1] 

机构地区：[1]华中师范大学生命科学学院,湖北武汉430079 [2]宜春学院医学院,江西宜春336000 [3]华中师范大学化学学院,湖北武汉430079

出　　处：《化学与生物工程》2007年第10期28-31,共4页Chemistry & Bioengineering

基　　金：国家重点基础研究发展规划项目(2003CB114400);国家自然科学基金资助项目(20372023)

摘　　要：为了在大肠杆菌中表达大肠杆菌丙酮酸脱氢酶,用PCR法从大肠杆菌总DNA中克隆了pdc基因,将其插入载体pGEX-KG后转化大肠杆菌BL21(DE3),在IPTG的诱导下实现了表达。通过对表达条件的优化,在TB+M9(1∶1)培养基中,33℃下使用终浓度0.5 g.L-1的乳糖诱导4 h,重组大肠杆菌丙酮酸脱氢酶的表达量可占全菌蛋白的45%左右,比未优化前提高了约20%,同时大大降低了成本。In order to express the E. coli pyruvate dehydrogenase in BL21 （DE3）, the gene pdc is amplified from E. coli total DNA. After digestion with SalⅠ and SacⅠ,the fragment is inserted into pGEX-KG, and the recombinant plasmid is named as pGEX-pdc. The recombinant plasmid is transformed into BL21（DE3）. In the inducement of IPTG, the recombinant proPDC is successfully expressed. According to use the different culture mediums, change the induction temperature and inducer concentration, use the lactose as inducer, and change the induction time,the recombinant proPDC can achieve about 45% of total protein in TB＋M9 culture medium, which is induced with 0. 5 g·L^-1 lactose at 33℃ for 4 h.

关 键 词：大肠杆菌丙酮酸脱氢酶 克隆 表达优化 

分 类 号：Q55[生物学—生物化学] Q786