The esterase III encoded by estC gene of Pseudomonas fluorescens was overexpressed in Escherichia coli BL21(DE3) by using the T7 expression system. The open reading frame of estC gene amplified by polymerase chain reaction was subcloned into T7 promoter-containing plasmid, pRSET. The resultant plasmid, pREIII was used for transformation of E. coli cells. The expression level of esteraseIII in E. coli harboring pREIII was 20 times higher than that harboring pUE892 which contains estC under lac promoter. The protein was successfully expressed without forming any inclusion body in side the E. coli cells. The amount of the enzyme was estimated to be 7-14\% of total cellular protein as judged by densitometer after SDS-gel electrophoresis. Following sequential DEAE-Sepharose ion exchange chromatography and Superose 12 gel filtration chromatography, the protein was purified to homogeneity. After purification, activity yield was 34\% and thr specific activity was 107000.
대장균에 cloning된 Peudomonas fluorescens SIK W1 균주의 estC 유전자가 encoding하는 esterase III를 대량 생산하기위해 T7 expression system을 이용하였다. estC gene의 open reading frame 을 증폭하기위해 PCR을 수행하였고, T7 promoter를 갖는 pRSET vector에 subcloning하였다. 결과적으로 만들어진 재조합 plasmid를 pREIII라고 명명하였고 대장균을 형질전환하는데 사용하였다. pREIII plasmid를 갖는 대장균의 esteraseIII 발현 수준은 lac promoter하에 estC를 가진 pUE892 plasmid를 가진 대장균에 비해 20배 증가하였다. pREIII로 부터 만들어지는 esteraseIII는 전체 세포 단백질의 7-14\% 정도로 추정되었다. DEAESepharose ion chromatpgrphy와 Superose12 gel filtration chromatography를 통해 순수하게 정제되었음이 SDS-PAGE를 통해 확인 되었다. 정제후에 activity yield는 34\% 였고, specific activity는 107000이었다.
