title: Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase.
abstract: Sacchromyces cerevisiae protein disulfide isomerase (yPDI) was expressed in the E. coli periplasm by using plasmids encoding the OmpA-yPDI-(His)(6) fusion gene under the control of the araBAD, trc, or T7 promoter. The expression levels of yeast PDI under these promoters were compared. Our results showed that yeast PDI expressed into the periplasm could catalyze the formation of disulfide bonds in alkaline phosphatase, restoring the phoA(+) phenotype in dsbA(-) mutants. The yeast PDI was purified from the Escherichia coli periplasm and shown to exhibit catalytic properties comparable to those of the rat enzyme with reduced RNase as substrate. In vivo, coexpression of the yeast PDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) in E. coli by 2-fold, similar to the effect seen previously with the coexpression of the rat enzyme. However yeast PDI was more effective than rat PDI in facilitating the expression of active tissue plasminogen activator (tPA). These results point to differences in the substrate specificity of various PDI enzymes, at least in the context of the E. coli periplasm.
authors.affiliation: Institute of Cell and Molecular Biology, Department of Chemical Engineering, University of Texas, Austin, Texas 78712, USA.
authors.forename: X
authors.initials: X
authors.lastname: Zhan
authors.forename: M
authors.initials: M
authors.lastname: Schwaller
authors.forename: H F
authors.initials: HF
authors.lastname: Gilbert
authors.forename: G
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000096:biosynthesis / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D004220:Disulfides; D011993:Recombinant Fusion Proteins; D007611:Aprotinin; D019704:Protein Disulfide-Isomerases