热玫瑰小双孢菌来源的丙酮酸磷酸双激酶的表达及应用  

Expression of PPDK from Microbispora rosea subsp,aerata in Escherichia.coli and Its Application in Pyrosequencing

作　　者：邹秉杰[1] 陈之遥[1] 周国华[1] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009

出　　处：《生物工程学报》2008年第4期679-683,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No．30470454);日本日立中央研究所资助~~

摘　　要：以热玫瑰小双孢菌基因组DNA为模板,通过PCR扩增得到了编码PPDK的基因,将此基因片段插入到表达载体pET28a(+)中构建得到了重组表达质粒pET28a(+)-PPDK,将重组表达质粒pET28a(+)-PPDK转化到大肠杆菌BL21(DE3)中,经过IPTG诱导,重组菌成功表达了N端带有6-His Tag的重组PPDK。经SDS-PAGE分析,重组PPDK单体分子量为101 kD。经过镍亲和层析和超滤后,重组PPDK蛋白基本达到电泳纯,并被成功应用于焦测序中。Pyruvate phosphate dikinase （PPDK; EC 2.7.9.1） is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate （PEP） to ATE Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp, aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET28a（＋）, yielding pET28a （＋）-PPDK. The E. coli BL21 （DE3） was transformed with the pET28a （＋）-PPDK. After inducing with IPTG, the E. coli BL21 （DE3） [pET28a （＋）-PPDK] expressed recombinant PPDK fused to an N-terminal sequence of 6-His Tag. The molecular weight of PPDK was estimated to be 101 kD by SDS-PAGE. The PPDK was purified by His · Bind Resin affinity chromatography and ultrafiltration using 10 kD cut-off membrane. The successful application of PPDK in pyrosequencing was also demonstrated.

关 键 词：PPDK 热玫瑰小双孢菌 大肠杆菌 焦测序 

分 类 号：Q78[生物学—分子生物学] S685.12[农业科学—观赏园艺]