Effects of macromolecular crowding on refolding of recombinant human brain-type creatine kinase

In this study, we quantitatively measured the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000), dextran 70, and calf thymus DNA (CT DNA), on the refolding and aggregation of recombinant human brain-type creatine kinase (rHBCK) denatured by guanidine hydrochloride (GdnHCl). The results showed that there is more aggregation in the presence of either a single crowding agent or in a mixture of crowding agents than in the absence of crowding agents, especially in the presence of a mixture containing CT DNA and PEG 2000 (or dextran 70). In the presence of high concentrations of PEG 2000 (100 g/L), dextran 70 (100 g/L), and CT DNA (15 g/L), the refolding yield remarkably decreased from 70% to 20%, 52% and 57%, respectively. A remarkable decrease in the refolding yield and rate with mixed crowding agent containing CT DNA and PEG 2000 (or dextran 70) was also observed. In comparison to refolding in the presence of 100 g/L PEG 2000, the refolding yields and rates improved in the presence of a mixture of PEG 2000 and dextran 70. We speculate that the crowding agents can favor both correct folding and misfolding/aggregation of denatured-rHBCK. Though it is not known what combination of crowding agents most accurately reflects the physiological environment within a cell, we believe our study could contribute to the understanding of protein folding and the factors that contribute to proper conformation and function in the intracellular environment.