2. Protein Refolding

Heterologous proteins produced by recombinant microbial organisms (e.g., E. coli.) often form inactive aggregates known as inclusion bodies. In order for the recovery of biologically active protein, the aggregates must be separated from cell debris and solubilized by exposing them to a strong denaturant. Decreasing the denaturant concentration by dialysis or direct dilution can initiate the unfolded polypeptide chains to refold to their native state. However, only a fraction of the native-state protein can be recovered in this process since the unfolded protein tends to re-aggregate by intermolecular interactions. This is because of the fact that protein renaturation is a kinetically competitive process between the folding and aggregation. As a unimolecular process, protein folding is a first-order reaction, while the aggregate formation is higher order because it is caused by intermolecular interactions. Due to the kinetic nature of protein refolding process, suppression of the aggregating reaction is the key point to enhance protein renaturation yield. Our research has focused on the efficient refolding of denatured-reduced proteins at high concentrations by various methods, including the application of molecular chaperones, artificial chaperones, chromatography, and fed-batch dilution. Mathematical models for the refolding processes have been established and the kinetic behaviors of the processes are evaluated. Currently, we devote to the design and discovery of protein folding enhancers that facilitate protein refolding at high concentrations. A recent development is about the finding of the facilitation effect of ion-exchange resins and polyelectrolytes on like-charged protein refolding by suppressing inter-molecular interactions that lead to protein aggregations (Biotechnol. Bioeng., 2011, 108: 1068). Studies of protein folding kinetics by using stopped-flow fluorescence spectroscopy have provided new insight into the molecular mechanism of arginine-assisted protein refolding.