[Over-expression of a polyketide synthase (PKS) module of a giant polyene antibiotic gene cluster in E. coli by double induction].
A 2,671 bp DNA carrying a type I PKS module with KS and AT domains from Streptomyces sp. FR-008 was cloned in-frame into the BamHI site immediately downstream of the PT7 promoter of the E. coli expression vector pET-15b, no considerable expression under IPTG induction was detected. The same PKS gene cloned downstream of the tandem PRPL promoters of pBV220 also yielded no over-expression under 42 degrees C induction. This gene was, however, over-expressed when it was cloned downstream of the tandem PRPT7 or PRPLPT7 promoters. In the case of the tandem PRPLPT7 promoters, the over-expression was dependent on the 42 degrees C plus IPTG double induction. While in the case of the tandem PRPT7 promoters, over-expression could be achieved when the gene was induced by IPTG or 42 degrees C individually or by IPTG and 42 degrees C double induction. Based on these experiences an expression vector pHZ330 containing the tandem PRPT7 promoters was constructed. In addition, the PKS protein expressed in E. coli was injected into rabbits to generate PKS-specific antibodies. Western blotting experiment indicated that these antibodies were PKS-specific which could be used either for the study of the PKS gene cluster or for the detection of the heterologous expression of Streptomyces sp. FR-008 PKS genes.
Tao
M F
MF
College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan.
Hu
Z H
ZH
Zhou
X F
XF
Zhou
Q
Q
Deng
Z X
ZX
Kieser
T
T
Hopwood
D A
DA
D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D009097:Multienzyme Complexes / Q000235:genetics* / Q000276:immunology; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D011817:Rabbits; D013302:Streptomyces / Q000235:genetics*
D009097:Multienzyme Complexes