Folding-like-refolding of heat-denatured MDH using unpurified ClpB and DnaKJE

Abstract

The Escherichia coli heat-shock protein ClpB can efficiently solubilize protein aggregates and refold them into active proteins in cooperation with the DnaK–DnaJ–GrpE chaperone (DnaKJE) system. However, the application of this bichaperone system at a large-scale was restricted because of the difficulties and high cost to express and purify each of these molecular chaperones. In this study, we constructed a plasmid encoding ClpB with a 6xHis-tag at its C-terminus (His-ClpB) to facilitate its purification through Immobilized Metal Affinity Chromatography (IMAC). A different plasmid capable of expressing the DnaKJE was used to obtain a cell extract containing unpurified DnaKJE. The effect of purified His-ClpB and unpurified DnaKJE on the refolding of heat-denatured malate dehydrogenase (MDH) was investigated, and proved to be highly efficient for MDH refolding. Furthermore, the use of both unpurified His-ClpB and DnaKJE available in the cell extract enabled highly successful refolding of the heat-denatured MDH with efficacy comparable to the case where the purified His-ClpB was used. To the best of our knowledge, this is the first attempt to apply a refolding cocktail comprising unpurified bichaperone system to the refolding of a heat-denatured protein, providing a practical and economically viable way of implementing a large-scale folding-like-refolding strategy.