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 scram bled ribonuclease assay, PDI activity was also demonstrated by complem entation of two phenotypes associated with a dsbA mutation, Alkaline p hosphatase activity, which is reduced in dsbA cells, was restored to w ild 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 it s activity to that seen in wild type cells. Increased expression of PD I was found to increase the yield of active PelC above that seen in wi ld type cells, PDI also enhanced the yield of PelC in DsbA(-) cells bu t only in the presence of exogenous oxidized glutathione, PDI is thus able to functionally substitute for DsbA in the folding of disulfide-b onded 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 a ctivities of DsbA and PDI in vivo may be different.