Overexpression and detection of the mutated glucose isomerase GIG138P and GIG138P-G247D in Streptomyces lividans / 生物工程学报

ABSTRACT

The shuttle expression vectors pHZGI1 and pHZGI2 were successfully constructed by inserting structural genes of GI containing single mutated site G138P and double mutated site G138P-G247D into E. coli-Streptomyces shuttle vector pHZ-1272, respectively. Then they were transformed into S. lividans TK54 strain by protoplast transformation. SDS-PAGE indicated that two shuttle vectors in TK54 strain expressed obviously specific bands at 42.5 kD after inducted by 2 micrograms/mL thiostrepton. Optical densitometric scan showed that the content of the mutant enzymes GIG138P and GIG138P-G247D were about 19% and 22% of dissoluble proteins, respectively. Western blotting farther proved that GIG138P and GIG138P-G247D were expressed in S. lividans TK54.