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

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 coli BL21(DE3) for effective expression,respectively.It can be proved that expression vectors of Y127F and R254F were successfully constructed.