【摘要】 通过三步亚克隆 ,将单点突变葡萄糖异构酶 ( GIG1 38P)基因及其调控序列插入链霉菌质粒p IJ40 83,构建重组表达质粒 p IJ40 83- GI1 .用重组质粒转化变铅青链霉菌 TK54原生质体 ,经硫链丝菌素抗性 ( Th R)筛选 ,获得重组菌株 TK54/p IJ40 83- GI1 .酶活力测定和 SDS- PAGE分析表明 ,GIG1 38P基因在变铅青链霉菌中得到高效表达 ,GI1粗酶液比活力为 1 5U/mg,GI1表达量约占菌体可溶性蛋白的 2 5% .同时也研究了重组质粒的遗传稳定性 .重组菌株在无选择压力条件下经液体连续传代培养 ,GI1比活力和 GI1表达量在 2 0 0 h传代时间中呈平缓下降趋势更多还原

【Abstract】 Single mutation glucose isomerase(GIG138P)gene and its natural promoter were isolated and inserted into Eco R Ⅰ Hin d Ⅲ linearized Streptomyces promoter probe plasmid vector pIJ4083 by three step subcloning.The ligation mixture was then introduced into Streptomyces lividans TK54 by protoplast transformation and the recombinant expression plasmid pIJ4083 GI1 was successfully obtained.Enzyme activity assay and SDS PAGE analysis indicated that the GIG138P(GI1)gene was overexpressed in S.lividans TK54,GI1 specific activity in crude extract was 15 U/mg and GI1 expressed accounted for nearly 25% of the soluble proteins in Streptomyces. The stability of recombinant plasmid in S.lividans was also determined.Continuous liquid cultures of recombinant strain under non selective conditions showed that GI1 specific activity and corresponding GI1 expressed in S.lividans decreased slightly within 200 hours.更多还原