Efficient in vitro refolding and functional characterization of recombinant human liver carboxtlesterases (CES1) expressed in E. coli
Issued Date
2014
Resource Type
Language
eng
Rights
Mahidol University
Suggested Citation
Usa Boonyuen, อุษา บุญยืน, Kamoltip Promnares, Suwapat Junkree, Day, Nichloas P.J., Mallika Imwong, มัลลิกา อิ่มวงศ์ (2014). Efficient in vitro refolding and functional characterization of recombinant human liver carboxtlesterases (CES1) expressed in E. coli. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/63186
Title
Efficient in vitro refolding and functional characterization of recombinant human liver carboxtlesterases (CES1) expressed in E. coli
Abstract
Human liver carboxylesterase 1 (CES1) plays a critical role in hydrolysis
of various ester- and amide-containing molecules, including active
metabolites, drugs and prodrugs. It has been problematic to express
recombinant CES1 in bacterial expression system. Due to low solubility, CES1 protein was mainly expressed in inclusion bodies, with insufficient purity. In this study, we reported an efficient in vitro method for refolding recombinant CES1 from inclusion bodies. One-step purification using immobilized metal affinity column was utilized to purify his-tagged recombinant CES1. Conveniently, denaturant and imidazole can
be removed while the enzyme is refolded via buffer exchanging, a dilution method. We showed that refolding recombinant CES1 was successful in Tris-HCl at pH 7.5 containing a combination of 1% glycerol and 2 mM b-mercaptoethanol. Mixtures of other additives (trehalose, sorbitol and sucrose) and b-mercaptoethanol failed to recover the functional protein. After refolding, his-tagged recombinant CES1 retained its biological activity and can be used directly without removing fusion tag. Taken together, our results provide an alternative method for obtaining substantial amount
of functionally active protein, which is advantageous for further investigations such as structural and functional studies.
Description
Joint International Tropical Medicine Meeting 2014: 3D perspectives on tropical medicine: drivers, diversity and determination the 8th seminar on food-and water-borne parasitic zoonoses: 2-4 December 2014: Centara Grand Bangkok Convention Center at Central World, Bangkok,Thailand. Bangkok: Faculty of Tropical Medicne, Mahidol University; 2014. p. 259.