Please use this identifier to cite or link to this item: https://doi.org/10.1016/j.bej.2007.11.026
Title: Folding-like-refolding of heat-denatured MDH using unpurified ClpB and DnaKJE
Authors: Nian, R.
Tan, L.
Choe, W.-S. 
Keywords: ClpB
DnaK-DnaJ-GrpE
MDH
Molecular chaperone
Protein aggregate
Refolding
Issue Date: 15-May-2008
Source: Nian, R., Tan, L., Choe, W.-S. (2008-05-15). Folding-like-refolding of heat-denatured MDH using unpurified ClpB and DnaKJE. Biochemical Engineering Journal 40 (1) : 35-43. ScholarBank@NUS Repository. https://doi.org/10.1016/j.bej.2007.11.026
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. © 2007 Elsevier B.V. All rights reserved.
Source Title: Biochemical Engineering Journal
URI: http://scholarbank.nus.edu.sg/handle/10635/63937
ISSN: 1369703X
DOI: 10.1016/j.bej.2007.11.026
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