title: Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli.
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 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.
authors.affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Electronic address: usa.boo@mahidol.ac.th.
authors.forename: Usa
authors.initials: U
authors.lastname: Boonyuen
authors.affiliation: Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkla 90112, Thailand. Electronic address: kamoltip.p@psu.ac.th.
authors.forename: Kamoltip
authors.initials: K
authors.lastname: Promnares
authors.affiliation: Central Equipment Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Electronic address: suwapat.jun@mahidol.ac.th.
authors.forename: Suwapat
authors.initials: S
authors.lastname: Junkree
authors.affiliation: Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, Oxford, United Kingdom. Electronic address: nickd@tropmedres.ac.
authors.forename: Nichloas P J
authors.initials: NP
authors.lastname: Day
authors.affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Electronic address: noi@tropmedres.ac.
authors.forename: Mallika
authors.initials: M
authors.lastname: Imwong
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D058849:Protein Refolding; D012995:Solubility
chemical_list: D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human
keywords: Carboxylesterases; E. coli; Glycerol; Inclusion bodies; Refolding