重叠延伸PCR技术构建β-环糊精葡糖基转移酶基因的定点突变原核表达载体

Site-directed mutagenesis of β-CGTase and expression vector construction by overlap extension PCR

采用重叠延伸PCR技术对环糊精葡萄糖基转移酶基因序列中保守区域的二个氨基酸位点Y127,R254进行体外基因定点突变。将突变基因分别亚克隆进pUC-18,经PCR鉴定及序列分析,所转化的Escherichia coli BL21(DE3)中含有插入的突变基因重组质粒,结果表明成功构建了Y127F、R254F二个突变基因的重组表达载体。

Using overlap extension PCR, two amino acid residues Y127 and R254 within the conserved protein domain of β-cyclodextrin glucosyl transferase were subjected to site-directed mutagenesis, respectively.The mutant genes were subcloned into prokaryotic expression vector pUC-18,by PCR and sequence analysis,these recombinant expression plasmids were transformed into Escherichia coil BL21 （DE3） for effective expression, respectively.It can be proved that expression vectors of Y127F and R254F were successfully constructed.