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Refolding of recombinant homodimeric malate dehydrogenase expressed in Escherichia coli as inclusion bodies

Paper ID Volume ID Publish Year Pages File Format Full-Text
4653 236 2008 8 PDF Available
Title
Refolding of recombinant homodimeric malate dehydrogenase expressed in Escherichia coli as inclusion bodies
Abstract

Escherichia coli malate dehydrogenase (eMDH) is a homodimeric enzyme composed of identical subunits, and the catalytically active form of the enzyme is a dimer. Herein, a recombinant eMDH was overexpressed in E. coli as inclusion bodies (IBs). The purified eMDH IBs were solubilized by urea for refolding studies. It was found that refolding at low temperature (4 °C) enhanced the refolding yield of the homodimeric enzyme. Some representative folding aids were examined, of which glycerol, sucrose and adenosine triphosphate were found to be effective. However, the refolding method with an artificial chaperone (AC) was the most efficient one that gave a specific activity recovery as high as 155 U/mg, about two times higher than a simple dilution. Analyses by high-performance size-exclusion chromatography, fluorescence spectroscopy and circular dichroism (CD) spectroscopy have well explained the reason for the high activity yield of the AC approach. That is, although the enzyme subunit was well folded to a tertiary structure close to its native state by both the simple dilution and the AC method, refolding by simple dilution led to less formation of dimeric protein, so its enzyme activity was much lower than that obtained by the AC method. CD spectra analysis indicates that there was higher α-helix fraction in the eMDH refolded by the AC method, and higher α-helix fraction is considered to be significant for the dimer formation of the enzyme.

Keywords
Malate dehydrogenase; Expression; Inclusion bodies; Refolding; Folding aids; Artificial chaperone
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Refolding of recombinant homodimeric malate dehydrogenase expressed in Escherichia coli as inclusion bodies
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 38, Issue 3, 15 March 2008, Pages 341–348
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