Construction and characterization of thermo-inducible vectors derived from heat-sensitive lacI genes in combination with the T7 A1 promoter

Biotechnology and Bioengineering
Yun-Peng ChaoHongyong Fu

Abstract

The lack of stringency and the cost of induction are two major disadvantages of using lac-derived vectors for recombinant protein productions. To compensate for these drawbacks, a series of thermo-inducible vectors was developed by coupling heat-sensitive lacI (lacIts) with the T7 A1 promoter on a multiple-copynumber plasmid. The lacIts genes were created by the introduction of Gly187-->Ser substitution along with three alternative mutation sites, Leu233-->Lys, Ala241-->Thr, and Gly265-->Asp, generated by site-directed mutagenesis into the wild-type lacI gene. With the LacZ production as a model, the induction profiles for various vectors containing distinct lacIts exhibited a positive trend as the temperature increased. The fully induced level was achieved by applying the temperature shift from 30 degrees C to 42, 40, or 37 degrees C to the cells harboring the plasmid with the Gly187-->Ser, Ala241-->Thr, or Gly265-->Asp substitution in lacI, respectively. As a result, it produced the maximal LacZ production ranging between 46,000 and 54,000 Miller units, corresponding to a 100- to 400-fold amplification over the uninduced level. As a whole, these novel expression vectors are characterized as having tight regulation and facile ...Continue Reading

References

Jun 25, 1979·Journal of Molecular Biology·J H Miller, U Schmeissner
Dec 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·M Lanzer, H Bujard
Dec 1, 1973·Proceedings of the National Academy of Sciences of the United States of America·K BeyreutherA Klemm
Jan 1, 1983·Methods in Enzymology·M RosenbergA Shatzman
Jan 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·H A de BoerM Vasser
Jun 14, 1995·Gene·K DhingraG N Hortobagyi
Jul 18, 1995·Proceedings of the National Academy of Sciences of the United States of America·G A WardT R Fuerst
Apr 11, 1993·Nucleic Acids Research·S Lisser, H Margalit

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Citations

Mar 8, 2011·Applied and Environmental Microbiology·K M McCabeM Hernandez
Jul 10, 2013·Microbial Cell Factories·Pietro Gatti-LafranconiFlorian Hollfelder
Feb 9, 2010·Pediatric Research·Kevin M McCabe, Mark Hernandez
Nov 15, 2016·Nature Chemical Biology·Dan I PiranerMikhail G Shapiro
Dec 3, 2009·Biotechnology Progress·Chung-Jen ChiangYun-Peng Chao
Dec 8, 2005·Biotechnology and Bioengineering·Sung Kuk Lee, Jay D Keasling

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