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

Paper ID Volume ID Publish Year Pages File Format Full-Text
4458 227 2008 9 PDF Available
Title
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.

Keywords
Molecular chaperone; ClpB; DnaK–DnaJ–GrpE; Protein aggregate; Refolding; MDH
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Folding-like-refolding of heat-denatured MDH using unpurified ClpB and DnaKJE
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 40, Issue 1, 15 May 2008, Pages 35–43
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us