Applicability of new expression vectors for both engineering uses and biological studies.
To be applicable for both engineering and biological uses, the plasmid with the features of tight regulation, high-level expression, and subtle modulation (or homogeneous induction) is required. IPTG-inducible promoters are of particular interest since they acquire the latter two merits but usually lack stringency. To this end, two plasmids have been developed to contain the T7 A1 promoter along with either lacI(q) or lacI gene. As a production system, the cells harboring the plasmids with the lacZ gene clone enabled production of the maximal protein accounting for 35% total cell content upon induction by a saturating IPTG level. This protein yield is amplified over 700-fold relative to that at the uninduced state. As a system for biological study, the ppc negative strain bearing the plasmid with the ppc gene clone failed to grow on glucose without IPTG induction but immediately resumed its growth in the presence of IPTG. Moreover, the level of the ppc gene product in the cell was varied by various IPTG, and the result revealed that the wild-type ppc level was sufficient to support the saturated growth of the cell on glucose. Overall, it illustrates the applicability of these plasmids to needs in the post-genome era.
Chao
Yun-Peng
YP
Department of Chemical Engineering, Feng Chia University, P. O. Box 25-102, 100 Wenhwa Road, Taichung, Taiwan. ypchao@fcu.edu.tw
Chiang
Chung-Jen
CJ
Wang
Yu-Ling
YL
Wang
Zei Wen
ZW
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*
D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside