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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/35584
Title: Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli
Authors: Usa Boonyuen
Kamoltip Promnares
Suwapat Junkree
Nichloas P.J. Day
Mallika Imwong
Mahidol University
Prince of Songkla University
Churchill Hospital
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Jan-2015
Citation: Protein Expression and Purification. Vol.107, (2015), 68-75
Abstract: © 2014 The Authors. Published by Elsevier Inc. Human liver carboxylesterase 1 (CES1) plays a critical role in the hydrolysis of various ester- and amide-containing molecules, including active metabolites, drugs and prodrugs. However, it has been problematic to express recombinant CES1 in bacterial expression systems due to low solubility, with the CES1 protein being mainly expressed in inclusion bodies, accompanied by insufficient purity issues. In this study, we report an efficient in vitro method for refolding recombinant CES1 from inclusion bodies. A one-step purification with an immobilized-metal affinity column was utilized to purify His-tagged recombinant CES1. Conveniently, both denaturant and imidazole can be removed while the enzyme is refolded via buffer exchange, a dilution method. We show that the refolding of recombinant CES1 was successful in Tris-HCl at pH 7.5 containing a combination of 1% glycerol and 2 mM β-mercaptoethanol, whereas a mixture of other additives (trehalose, sorbitol and sucrose) and β-mercaptoethanol failed to recover a functional protein. His-tagged recombinant CES1 retains its biological activity after refolding and can be used directly without removing the fusion tag. Altogether, our results provide an alternative method for obtaining a substantial amount of functionally active protein, which is advantageous for further investigations such as structural and functional studies.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84918521617&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/35584
ISSN: 10465928
Appears in Collections:Scopus 2011-2015

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