Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds.
Eukaryotic protein disulfide isomerase (PDI) is a 55-kDa enzyme with cysteine oxidoreductase, chaperone, and antichaperone activities that catalyzes disulfide formation and rearrangement in the eukaryotic endoplasmic reticulum. In Gram-negative bacteria, the formation of disulfide bonds in the periplasm is mediated by DsbA, a strong cysteine oxidase but an inefficient catalyst of disulfide bond isomerization with no known chaperone activity. We show that rat PDI (rPDI) secreted in the periplasmic space of Escherichia coli can catalyze the formation of disulfide bonds and complement several of the phenotypes of dsbA mutants. The function of rPDI was dependent on the dsbB gene, suggesting that the reoxidation of this eukaryotic enzyme involves direct interactions with bacterial redox proteins. Co-expression of rPDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) severalfold, an effect that was enhanced when reduced glutathione was added to the growth medium. Whereas PDI is thought to function primarily as an isomerase in the eukaryotic endoplasmic reticulum, rPDI failed to decrease the accumulation of two-disulfide folding intermediates of BPTI and thus did not appear to appreciably catalyze the rate-limiting step in the oxidative folding pathway of BPTI. These results demonstrate that expression of eukaryotic foldases in E. coli can be exploited to better understand their function in vivo and also to increase the yield of biotechnologically valuable proteins.
Ostermeier
M
M
Department of Chemical Engineering, University of Texas, Austin 78712, USA.
De Sutter
K
K
Georgiou
G
G
D000818:Animals; D007611:Aprotinin / Q000235:genetics; D002384:Catalysis; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism; D010641:Phenotype; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D051381:Rats
D004220:Disulfides; D007611:Aprotinin; D010088:Oxidoreductases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases