Genetic and biochemical analysis of disulfide bond isomerization in Escherichia coli

The isomerization of disulfide bonds in the Escherichia coli periplasm was studied using human tissue plasminogen activator (tPA) as a model protein substrate.; tPA was secreted into the periplasm by using the E. coli heat-stable enterotoxin signal sequence. The yield of active tPA in bacteria was shown to be extremely low in wild type cells. The rate-limiting step in the folding of tPA was shown to be related to the formation of the 17 disulfide bonds that are present in the native conformation of the protein. The effect of co-expression of native (DsbA and DsbC) and heterologous (rat protein disulfide bond isomerase, rPDI) cysteine oxidoreductases was systematically investigated.; A fibrin overlay agar plate assay was developed to visualize tPA fibrinolytic activity in situ without recourse to cell fractionation. An E. coli genomic library was then co-expressed with tPA to identify novel genes that enhanced tPA activity, most likely by promoting disulfide bond isomerization. After the screening of 10,000 colonies, twelve clones showing enhanced activity were identified. Three of these genes, menG, yjgD, and yhbL, that were either poorly characterized or had unknown function were studied further.; The menG gene encodes a 17.4 kDa protein and had been previously assigned a methyltransferase function. MenG overexpression resulted in hyperoxidation of DsbA in the periplasm.; Two other genes identified in the screen were yjgD and yhbL. YjgD is a putative oxidoreductase and gave similar phenotypes as menG when over expressed. YhbL encodes a highly conserved protein SCRP-27A (s&barbelow;igma c&barbelow;ross r&barbelow;eacting p&barbelow;rotein) that exhibits immunochemical cross-reactivity with σ70 and σ32. Both proteins were overexpressed using the pET system and purified using anion exchange chromatography to enable structural studies to be initiated.; In conclusion, this work demonstrated the following: (i) high level coexpression of the bacterial disulfide isomerase DsbC is necessary in order to produce active, heterologous proteins with multiple disulfide bonds in bacteria; (ii) a novel genetic screen for the identification of loci that affect disulfide bond formation was developed; (iii) the phenotypes conferred by one of the genes identified in the screen, MenG, were studies in some detail; and finally, (iv) the polypeptide products of two genes isolated by the screen were purified to homogeneity in preparation of X-ray crystallography. (Abstract shortened by UMI.)...