Improvement of the thermoregulated T7 expression system by using the heat-sensitive lacI

Biotechnol Prog. Sep-Oct 2004;20(5):1352-8. doi: 10.1021/bp049851r.

Abstract

The thermoregulated T7 expression system was previously reported to be an effective way to produce massive amounts of recombinant proteins (Chao, Y. P.; Law, W. S.; Chen, P. T.; Hung, W. B. High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system. Appl. Microbiol. Biotechnol. 2002b, 58, 446-453). To ensure its practical applicability, the system was improved for stringency with the construction of the T7lac-promoter-containing plasmid associated with the thermolabile lacI gene (lacIts). Owing to the recessive feature of lacIts, the wild-type lacI was removed from the genome of the cell. Moreover, the cell was engineered to carry the chromosomal copy of the T7 gene 1 subject to the regulation of lambdaPL and lambdaPR promoters. To characterize the system, the lacZ gene was fused to the T7lac promoter, and subsequent experiments showed that various amounts of LacZ could be synthesized in the plasmid-bearing cell in response to heat. Among the producers, the cell with the plasmid containing lacIts (substitution of Gly265 with Asp in lacI) was able to produce the maximal LacZ, the production accounting for an amplification of more than 200-fold over the uninduced level. A further demonstration was carried out to illustrate the practical usefulness of the developed system by producing carbamoylase on a 4000 L scale. Cultured to reach high cell density, the carbamolyase-producing cell was shown to retain plasmids with 95% stability and to be capable of producing soluble protein equal to 13% of the total cell proteins. Overall, it illustrates the remarkable features of the developed system with tightness, high expression level, thermal inducibility, and high stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / biosynthesis*
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Bacteriophage T7 / genetics*
  • Bioreactors / microbiology
  • Cloning, Molecular / methods*
  • DNA-Directed RNA Polymerases / genetics
  • DNA-Directed RNA Polymerases / metabolism
  • Gene Expression Regulation, Bacterial / physiology
  • Gene Expression Regulation, Enzymologic / physiology
  • Genetic Enhancement / methods*
  • Hot Temperature
  • Industrial Microbiology / methods
  • Lac Repressors
  • Promoter Regions, Genetic / genetics
  • Protein Engineering / methods*
  • Recombinant Proteins / biosynthesis
  • Repressor Proteins / genetics*
  • Repressor Proteins / metabolism*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • beta-Galactosidase / biosynthesis*
  • beta-Galactosidase / genetics

Substances

  • Bacterial Proteins
  • Lac Repressors
  • Recombinant Proteins
  • Repressor Proteins
  • Viral Proteins
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • beta-Galactosidase
  • Amidohydrolases
  • dihydropyrimidinase