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Title: Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli.
Authors: Usa Boonyuen
อุษา บุญยืน
Kamoltip Promnares
กมลทิพย์ พรหมณเรศ
Suwapat Junkree
Day, Nichloas P.J.
Mallika Imwong
มัลลิกา อิ่มวงศ์
Mahidol University. Faculty of Tropical Medicine. Mahidol Oxford Research Unit.
Mahidol University. Faculty of Tropical Medicine. Central Equipment Unit.
Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.
Mallika Imwong
Keywords: Carboxylesterases;E. coli;Glycerol;Inclusion bodies;Refolding;Open Access article
Issue Date: Mar-2015
Citation: Boonyuen U, Promnares K, Junkree S, Day NP, Imwong M. Efficient in vitro carboxylesterase (CES1) expressed in E. coli. Protein Expr Purif. 2015 Mar;107:68-75.
Abstract: 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 2mM 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.
ISSN: 1046-5928 (printed)
1096-0279 (electronic)
Appears in Collections:TM-Article

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