earticle

영어

High-level expression of recombinant proteins in E. coli often results in accumulation of insoluble aggregates known as inclusion bodies (IBs), requiring further solubilizing and refolding procedures prior to obtaining functionally active products. Although molecular chaperones have been harnessed with some success to refold proteins, the exact function of individual chaperones and the interactions between target protein and chaperones are still unclear. We demonstrated that a refolding cocktail comprising ClpB/DnaKJE, PEG and ATP regeneration system could significantly enhance the refolding efficiency of heat-denatured malate dehyrogenase (MDH).[1][2] To further clarify the individual or synergistic roles of each chaperone, diverse His-tagged molecular chaperones were cloned, expressed and purified, respectively. Heat treatment of MDH at different temperatures resulted in protein aggregates of varying sizes. Following the characterization of the protein aggregates for susceptibility to proteolysis, chaperoning efficiencies on the solubilization and renaturation of the protein aggregates have been studied. Various ratios of chaperones were applied with ATP regeneration system to refold MDH to determine an optimum condition of refolding cocktail.