Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli

Usa Boonyuen,Kamoltip Promnares,Suwapat Junkree,Nichloas P.J Day,Mallika Imwong

doi:10.1016/j.pep.2014.11.006

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.

Highlights

Human liver carboxylesterase (CES1, EC number: 3.1.1.1, 3.1.1.56), an enzyme responsible for the hydrolysis of ester- and amide-containing molecules, is mainly expressed in the liver where it is crucial for the processing of active metabolites and is involved in trans-esterification reactions [1,2,3,4]
We report the heterologous expression of carboxylesterase 1 (CES1) in E. coli, in which it was mainly expressed in inclusion bodies
The reaction mixture containing 930 ll of 50 mM Tris–HCl, pH 7.5, 40 ll absolute ethanol and 10 ll of various concentrations of pnitrophenyl acetate (pNPA) was incubated at 37 °C for 5 min

Summary

Introduction

Human liver carboxylesterase (CES1, EC number: 3.1.1.1, 3.1.1.56), an enzyme responsible for the hydrolysis of ester- and amide-containing molecules, is mainly expressed in the liver where it is crucial for the processing of active metabolites (heroin and cocaine) and is involved in trans-esterification reactions [1,2,3,4]. CES1 is known to play an important role in the hydrolysis of several classes of drugs and the activation of prodrugs, including angiotensin-converting enzyme (ACE) inhibitors and the anti-influenza agent oseltamivir [5,6,7,8,9,10], known as Tamiflu, a recommended treatment by the World Health Organization (WHO). Two known CES1 mutations, G143E and p.D260fs, have been shown to impair the function of the enzyme with regard to the activation of oseltamivir, indicating the significant role of CES1 in oseltamivir metabolism [8,9]. Extensive studies have been carried out to investigate the role of CES1 in oseltamivir metabolism in terms of pharmacokinetics, structure and function.

Methods

Results

Discussion

Conclusion