Human protein disulfide isomerase functionally complements a dsbA mutation and enhances the yield of pectate lyase C in Escherichia coli.
Human PDI was expressed to the Escherichia coli periplasm, by using a plasmid encoded ompA-PDI fusion under the control of the trp promoter. Periplasmic extracts were shown to contain active PDI using the scrambled ribonuclease assay. PDI activity was also demonstrated by complementation of two phenotypes associated with a dsbA mutation. Alkaline phosphatase activity, which is reduced in dsbA cells, was restored to wild type levels by PDI. PelC, a pectate lyase from Erwinia carotovora, was shown to be DsbA dependent in E. coli. PDI was able to restore its activity to that seen in wild type cells. Increased expression of PDI was found to increase the yield of active PelC above that seen in wild type cells. PDI also enhanced the yield of PelC in DsbA- cells but only in the presence of exogenous oxidized glutathione. PDI is thus able to functionally substitute for DsbA in the folding of disulfide-bonded proteins in the bacterial periplasm and to enhance the yield of highly expressed protein when the ability of the E. coli periplasm to fold protein may be saturated. However, our results suggest that the activities of DsbA and PDI in vivo may be different.
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D004885:Erwinia / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis*; D007535:Isomerases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000096:biosynthesis*; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases