Development and application of small molecule chaperones for protein renaturation

Protein aggregation presents a major problem in protein renaturation. This is due to competing intramolecular and intermolecular processes in unfolded protein molecules. Current investigations in our lab have focused on examining additives that can aid denatured polypeptide chains to refold into their native conformations and stabilize the structure of resulting proteins. Our approach to this problem is the development of a series of fluorous and non-fluorous salt additives that are capable of stabilizing protein structure against irreversible thermal denaturation, allowing protein molecules to refold into their native conformations, and recovering protein activities after chemical denaturation. Our initial studies employed Hen Egg White Lysozyme (HEWL) and Carbonic Anhydrase B (CAB) as model proteins to explore the effects of these salts on protein stabilities and renaturation. We also studied inclusion bodies of MMP 13 provided by Pfizer, Inc, and examined the utility of our protocol in a more practical application.

The behavior of fluorous and non-fluorous salts prepared in our lab was investigated in the case of both thermal and chemical denaturation of HEWL and CAB. Differential Scanning Calorimetry (DSC) was employed to monitor the thermal process of protein solutions treated with salt additives. While many of those salts are able to prevent aggregation of HEWL during heating at a high temperature, fluorous salts were found to have an unusual stabilizing effect on protein structure. Moreover, compared with non-fluorous salts, fluorous salts could generate higher recovered enzymatic activities from chemically reduced-denatured HEWL at a useful concentration. The structure of recovered proteins was investigated further using Circular Dichroism (CD) spectroscopy. Although difficulties were encountered in attempting to prevent the irreversible thermal denaturation of CAB, fluorous salts have significantly enhanced the recovery of chemically denatured CAB molecules at a relatively high concentration. In our attempt to renature MMP 13 inclusion bodies, we obtained the results that further confirm the critical importance of the fluorous character in salt additives, hi addition, all the refolded-active proteins were separated from the salts by a simple dialysis protocol.

Our approach to protein renaturation has a number of advantages. Furthermore, the observation of the important role of fluorous portion in stabilizing and recovering native protein structure provides the potential to develop a series of highly efficient additives for protein renaturation.