Kinetic study of recombinant human protein disulphide isomerase-assisted C125A recombinant human interleukin-2...

A kinetic model was developed to describe recombinant human protein disulphide isomerase (rhPDI)-assisted folding of a substrate protein, C125A recombinant human interleukin-2 (C125A rhIL-2). A series of progress curves showing native C125A rhIL-2 formation under different reaction conditions were generated. Non-linear regression analysis of the progress curves of rhPDI-assisted C125A rhIL-2 folding was used to fit the differential equations of the described kinetic models. The goodness-of-fit of the model to the experimental datasets was used to support or exclude a particular kinetic model of rhPDI-assisted C125A rhIL-2 folding. The results suggest that the formation of native C125A rhIL-2 results from both glutathione-dependent oxidative folding and rhPDI-catalysed folding reactions. During oxidative folding of C125A rhIL-2, both rhPDI and reduced C125A rhIL-2 aggregated in folding buffer. The aggregation rates of rhPDI and C125A rhIL-2 followed second-order kinetics. Guanidinium chloride inactivated rhPDI but also decreased the aggregation of reduced C125A rhIL-2. These results demonstrate that during rhPDI-assisted C125A rhIL-2 folding, reduced C125A rhIL-2 aggregation competes with the productive folding pathway. While rhPDI enhances the oxidative folding of C125A rhIL-2, inactivation of rhPDI by the residual guanidinium chloride compromises its catalytic efficiency. The established model can be used to optimize the folding components in the folding mixture, and thus improve the folding efficiency.