Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability

Gene. 1998 Mar 16;209(1-2):95-103. doi: 10.1016/s0378-1119(98)00020-1.

Abstract

E. coli recombinant expression systems that utilize lac operon control elements to modulate gene expression are known to produce some amount of uninduced (leaky) gene expression. Previously, we showed that high levels of uninduced gene expression was a major cause of instability in the pET expression system. We show here that the pET system, in which the phage T7 RNA polymerase gene is expressed via lac operon control elements, exhibits leaky expression that increases markedly as cells grown in complex medium enter stationary phase. Moreover, we found that this phenomenon occurs with the chromosomal lac operon as well. Further investigation revealed that stationary phase leaky expression requires cyclic AMP, and that substantial leaky expression could be effected in log phase cells by adding cyclic AMP and acetate at pH6.0. Finally, a comparison of otherwise isogenic cya and wild-type hosts showed that expression stability and plasmid maintenance in the cya host is greatly enhanced, even when cells are passaged repeatedly in non-selection medium. These findings both provide a method to enhance the stability of lac-based recombinant expression systems, and suggest that derepression of the lac operon in the absence of inducer may be part of a general cellular response to nutrient limitation.

MeSH terms

  • Bacteriophage T7 / enzymology
  • Bacteriophage T7 / genetics
  • Chromosomes, Bacterial
  • Cloning, Molecular / drug effects
  • Cloning, Molecular / methods*
  • Cyclic AMP / pharmacology*
  • DNA-Directed RNA Polymerases / biosynthesis*
  • DNA-Directed RNA Polymerases / genetics
  • Escherichia coli / drug effects
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Gene Expression Regulation, Bacterial*
  • Kinetics
  • Lac Operon*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Proteins / biosynthesis
  • Recombination, Genetic
  • Viral Proteins
  • beta-Galactosidase / biosynthesis*

Substances

  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Viral Proteins
  • Cyclic AMP
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • beta-Galactosidase