Approaches to high-performance protein refolding

Protein refolding from inclusion bodies is a crucial step in the downstream processing of therapeutic proteins. Because in vitro protein refolding operation is a kinetically competitive process between folding and aggregation, accelerating on-pathway folding and/or suppressing aggregation are effective approaches to high-performance protein refolding. Here, three examples of protein refolding methods are described, including (1) on-column refolding by artificial chaperone-assisted immobilized metal affinity chromatography that gives rise to simultaneous protein refolding and purification, (2) suppression of aggregation with like-charged ion exchange resin by electrostatic repulsion that leads to the significant increase of refolding yield, and (3) design of small-molecular foldase mimics that improves protein refolding yield by accelerating disulfide formation and reshuffling.