Effect Of Osmolytes On Protein Folding/Refolding And Stabilization

This thesis is concerned with an extensive study of protein stability, folding and molecular interaction in the presence of osmolytes, betaine and trehalose. The purpose of the research is to give clearer insight into the role of the osmolytes in the refolding or renaturation of proteins to be recovered from inclusion bodies.At first, guanidinium chloride-induced unfolding of lysozyme in the presence of betaine and trehalose were studied by tryptophan flurescence spectroscopy. Equilibrium stability parameters of lysozyme unfolding were obtained using the linear extrapolation model. It was found that the free energy of unfolding was enhanced by increasing the concentrations of both the osmolytes, resulting in the thermodynamic stabilization of lysozyme.Second, betaine and trehalose were used as folding aids to assist the renaturation of denatured-reduced lysozyme. It was observed that the refolding yield was improved with the increase in betaine concentration up to 2 mol·L-1, while it appeared to present an optimal trehalose concentration at about 0.2 mol·L-1. Then, the kinetic behavior of the refolding process was studied at different guanidinium chloride concentrations, and the dynamic process was expressed by the competitive model of first-order folding reaction and third-order aggregation. In the presence of betaine, the refolding rate was accelerated, leading to the increase of the refolding yield. The unfolded proteins could be stabilized by trehalose, and the aggregation of lysozyme was suppressed at low trehalose concentrations (<0.2 mol·L-1), thus facilitating the refolding. However, lysozyme renaturation was inhibited at high trehalose concentrations (>0.2 mol·L-1), showing low folding rates revealed by the kinetic model.Third, to reveal the effects of the osmolytes on the intermolecular interactions during protein refolding, self-interaction chromatography was employed to study the effect of betaine, trehalose and arginine on the second virial coefficient, BB22, of lysozyme in solutions containing guanidinium chloride or NaCl at different pH values. At all guanidinium chloride concentrations (0~6 mol·L- 1), B22 increased by raising betaine and trehalose concentrations. With increasing arginine concentrations, B22 decreased at low guanidinium chloride concentrations, but increased at high guanidinium chloride concentrations. The results indicated the different effects of the osmolytes from that of arginine in the intermolecular interactions.Finally, recombinant E. coli malate dehydrogenase (eMDH) was expressed as inclusion bodies (IBs), and the eMDH IBs was used as a real oligomeric protein to study protein refolding from IBs. It was observed that the specific activity of eMDH was enhanced by increasing betaine concentration, while there was an optimal trehalose concentration (0.5 mol·L-1) at which the protein was favorably renatured. Furthermore, the renaturation could be improved even at high guanidinium chloride concentrations (up to 0.4 mol·L-1) in the presence of betaine or trehalose.