Publication: Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli.
Issued Date
2015-03
Copyright Date
2014
Resource Type
Language
eng
ISSN
1046-5928 (printed)
1096-0279 (electronic)
1096-0279 (electronic)
Rights
Mahidol University
Rights Holder(s)
Elsevier Open Access
Bibliographic Citation
Protein Expression and Purification. Vol.107, (2015), 68-75
Suggested Citation
Usa Boonyuen, อุษา บุญยืน, Kamoltip Promnares, กมลทิพย์ พรหมณเรศ, Suwapat Junkree, Day, Nichloas P.J., Mallika Imwong, มัลลิกา อิ่มวงศ์ Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli.. Protein Expression and Purification. Vol.107, (2015), 68-75. doi:10.1016/j.pep.2014.11.006. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/847
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Title
Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli.
Corresponding Author(s)
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.