Development of a thermo-regulated expression vector in Escherichia coli B strain

Abstract

The lacI gene in the plasmid bearing the T7A1 promoter (PA1)-driven lacZ was randomly mutated. The mutant library was then screened in Escherichia coli B strain deficient in lacI and lacZ. Based on the LacZ phenotype, one heat-sensitive lacI (lacIts) was isolated and it revealed a mutation with an amino acid substitution, Met42Lys (designated lacI42ts). To examine its performance, the lacI42ts/PA1-based plasmid was employed for expression of gehC (encoding lipase) in E. coli B strain. Consequently, the strain that received a thermal induction produced 49-fold more GehC in terms of activity than the uninduced level. The expression condition was further optimized, finally leading to a 47% increase in the GehC activity for the strain. Overall, it indicates that the thermo-regulated vector is useful for the recombinant protein production in E. coli B strain.