Effects of Protein Disulfide-isomerase on the Refolding of Human Pro-urokinase Cloned and Expressed in Escherichia coli

Abstract

Protein disulfide-isomerase (PDI) was studied for the first time as a stimulator of refolding of recombinant proteins produced in bacteria. Experiments with reduced or scrambled ribonuclease as a substrate showed that PDI was active in low concentrations of the protein denaturants which were generally used in the refolding systems of recombinant proteins. Crude human pro-urokinase cloned and expressed in Escherichia coli was tested as an example of a recombinant protein, and PDI accelerated its refolding. The time needed to reach 50% maximum pro-urokinase refolding was shortened by half by the addition of PDI. Reduced pro-urokinase was further stimulated to refold by this enzyme. Evidence that PDI catalyzes the refolding of pro-urokinase through thiol-disulfide exchange reactions was also obtained from the repression of such stimulation by a PDI inhibitor, bacitracin. The results suggested that this refolding system can be used for the refolding of recombinant proteins produced in bacteria.