枯草芽孢杆菌ATCC21216腺苷磷酸化酶基因的克隆及其在大肠杆菌中的表达

Cloning and Expression of Bacillus subtilis ATCC21216 Adenosine Phosphorylase Gene in Escherichia coli

摘要: 聚合酶链式反应(PCR)扩增枯草芽孢杆菌ATCC21216(His-)编码腺苷磷酸化酶的基因deoD(0.7 kb),测序表明与枯草芽孢杆菌168的deoD同源性为98.7%。将此基因插入质粒pET28a( )(5.3 kb)中,重组质粒pETdeoD在大肠杆菌BL21(DE3)中实现了表达,表达量占总蛋白的27%。以HPLC方法测定腺苷磷酸化酶的比酶活为每毫克蛋白0.151 U。

Abstract: The gene deoD(0.7 kb) of B.subtilis ATCC21216 encoding an adenosine phosphorylase was amplified by polymerase chain reaction(PCR).Results of sequencing showed 98.7% similarity to the same gene of B.subtilis 168.Based on DNA recombinant techniques,the gene deoD was cloned into plasmid pET28a( )(5.3 kb) to generate highly effective E.coli BL21(DE3) strain producing adenosine phosphorylase.Expression of the recombinant protein was 27% of the total protein.Specific activity of the(enzyme) in crude extract determined by high performance liquid chromatography was 0.151 U/mg protein.