The molecular chaperone Skp reduces client protein aggregation under in vivo-like conditions

Abstract

Recombinant proteins form the basis of a vibrant biotechnology industry, acting as key players in therapeutics, reagents and diagnostics.1 However, multi-chain proteins are hard to produce efficiently.1 The yield of recombinant proteins can be improved by co-expression of folding factors with the target protein.2 Co-expression with folding factors has been met with some success.2 However, deeper knowledge about the folding factors and the ways in which they interact with each other will ultimately improve expression of proteins. The bacterial seventeen kilodalton protein (Skp) has been exploited to prevent aggregation of recombinant proteins in the bacterial periplasm.3 While the interactions between Skp and its client proteins have been studied in vitro, the more relevant in vivo interactions have not, nor has its relationship to the other protein folding molecules in the bacterial periplasm. In particular, the order of interactions between Skp and the Disulfide bond isomerases A and C (DsbA, DsbC) is unclear. To address this knowledge gap, the effect of Skp on substrate protein aggregation was observed under in vivo-like conditions as a step towards analyzing the effect of Skp in vivo. The effect of Skp on aggregation was reduced under in vivo-like conditions as compared to dilute in vitro experiments. The order of interaction between Skp, DsbA and DsbC on a substrate protein was observed. In the first experiment, the effect of the combination of Skp with DsbA and Skp with DsbC on the aggregation of lysozyme was observed. The greatest reduction in the aggregation of lysozyme was due to the addition of Skp with either DsbA or DsbC. In the second experiment, the order of interaction between Skp, DsbA and DsbC was assayed but no clear conclusion could be drawn. These insights will contribute to the understanding of protein expression in the bacterial periplasm.