GONUTS has been updated to MW1.31 Most things seem to be working but be sure to report problems.
PMID:25462813
| Citation |
Boonyuen, U, Promnares, K, Junkree, S, Day, NP and Imwong, M (2015) Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli. Protein Expr. Purif. 107:68-75 |
|---|---|
| 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. |
| Links |
PubMed PMC4294421 Online version:10.1016/j.pep.2014.11.006 |
| Keywords |
|
| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0016787: hydrolase activity |
ECO:0000314: |
F |
This paper looks at and tests the functions of liver carboxylesterase 1 which includes necessary information for the annotation |
complete | ||||
Notes
See also
References
See Help:References for how to manage references in GONUTS.
