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.

Keywords: Carboxylesterases; E. coli; Glycerol; Inclusion bodies; Refolding.

Fig. 1

Amino acid sequence of CES1 isoform a. The first 19-amino acids, highlighted in gray, comprise the signal peptide sequence, which was omitted during cloning. Amino acids known to be associated with the biological function are boxed. Natural variants created in our experiment are underlined. The N-glycosylation site is circled, and disulfide bonds are shown by linkage of two cysteine residues.

Fig. 2

SDS–PAGE analysis of recombinant WT CES1 expressed at different temperatures. (A) Cells were grown at 37 °C, induced with 0.5 mM IPTG and incubated at 25 °C for 4 h before harvest. Lane M, molecular mass marker proteins; lane 1, uninduced cells; lane 2, induced cells; lane 3, inclusion bodies and lane 4, soluble fraction. (B) Cells were grown at 25 °C and induced with 0.5 mM IPTG; lane 1, uninduced cells; lane 2, induced cells; lane 3, inclusion bodies and lane 4, soluble fraction. The arrow indicates the expected size of recombinant CES1.

Fig. 3

SDS–PAGE analysis of recombinant WT CES1 expressed at 25 °C in the presence of additives in the growth medium. (A) Final concentration of 1% glycerol at the time of induction; Lane M, molecular mass marker proteins; lane 1, uninduced cells; lane 2, induced cells; lane 3, inclusion bodies and lane 4, soluble fraction. (B) A final concentration of 0.2 M sorbitol was added to growth medium at the time of induction; lane 1, uninduced cells; lane 2, induced cells; lane 3, inclusion bodies and lane 4, soluble fraction. The arrow indicates the expected size of recombinant CES1.

Fig. 4

SDS–PAGE analysis of purified recombinant CES1s, WT, S76N, D204E and A270S, after refolding in 50 mM Tris–HCl, pH 7.5, containing 1% glycerol and 2 mM β-mercaptoethanol.

Fig. 5

Intrinsic fluorescence emission spectra of refolded CES1s, WT, S76N, D204E and A270S. Protein refolding was performed in 50 mM Tris–HCl, pH 7.5, containing 1% glycerol and 2 mM β-mercaptoethanol.

Fig. 6

Circular dichroism (CD) spectra of refolded CES1s, WT, S76N, D204E and A270S. Protein refolding was performed in 50 mM Tris–HCl, pH 7.5, containing 1% glycerol and 2 mM β-mercaptoethanol.

Fig. 7

A representative time course of pNPA hydrolysis by WT CES1. The reaction was performed in a 1 ml volume containing Tris–HCl, pH 7.5, absolute ethanol, 100 mM pNPA and 20 μl purified WT CES1 at 37 °C. Hydrolysis was determined after incubating the reaction at 37 °C for 10 min, and the formation of pNP was followed at 405 nm for 10 min. A blank reaction was performed by replacing the enzyme with buffer.

Fig. 8

A representative Michaelis–Menten plot of WT CES1. Enzyme kinetics of pNPA hydrolysis catalyzed by WT CES1. The initial velocity was obtained by subtracting the blank hydrolysis from the enzymatic hydrolysis at each substrate concentration.

Fig. 9

Western blot analysis of purified 6×His recombinant CES1, WT, S76N, D204E and A270S after refolding in 50 mM Tris–HCl, pH 7.5, containing 1% glycerol and 2 mM β-mercaptoethanol. Proteins were immunodetected using an anti-His (N-term) antibody.