Genetic and biochemical studies of disulfide bond isomerization in Escherichia coli

Heterologous proteins containing multiple disulfide bonds cannot fold efficiently when expressed in secreted form in Escherichia coli bacteria. The rate-limiting step in the folding of multidisulfide proteins in bacteria is the rearrangement of incorrect disulfide bonds, a process catalyzed by disulfide isomerases. Two strategies for enhancing disulfide bond isomerization in E. coli were investigated: (i) Co-expression of eukaryotic disulfide isomerases; (ii) Isolation of chromosomal lesions that improve the folding of the complex eukaryotic enzyme tissue plasminogen activator (tPA) which contains 17 disulfide bonds. The co-expression of the yeast protein disulfide isomerase (PDI) was found to increase the yield of native bovine pancreatic trypsin inhibitor by 2-fold, similar to the effect previously seen with the co-expression of the rat PDI. It was also shown that co-expression of yeast PDI was more effective than that of rat PDI in facilitating the expression of active tPA in E. coli periplasm. A novel genetic screen was designed and used to identify E. coli mutants that enhanced the production of active tPA. Nine mutant strains that exhibited significantly higher disulfide isomerization activity were isolated. All mutants showed markedly elevated DsbC protein expression. Unexpectedly, in eight out of the nine mutants, the upregulation of DsbC was mediated by defects in RNA processing by RNase E which is encoded by the rne gene: 5/9 mutations were allelic to rne whereas 3/9 were shown to exhibit impaired RNase E activity due to lesions in other loci. The importance in mRNA processing on the expression of DsbC is underscored by the half-life of the dsbC mRNA which is only 0.8 minutes at 37o C. The E.coli endonuclease RNase E is a 1061 amino acid protein that plays a dominant role in RNA degradation. RNase E consists of an N-terminal catalytic domain and a C-terminal half (CTH) region. The latter region serves as a scaffold for the binding of various proteins. It was found that MenG, which increases E. coli disulfide isomerization when it is overexpressed, negatively regulates RNase E enzymatic activity. It was further shown that this regulation depends on the presence of the RNase E CTH region and involves MenG-RNase E interaction.