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Ligand-enhanced expression and in-cell assay of human peroxisome proliferator-activated receptor alpha ligand binding domain

Velkov, Tony, Rimmer, Kieran A. and Headey, Stephen J. 2010, Ligand-enhanced expression and in-cell assay of human peroxisome proliferator-activated receptor alpha ligand binding domain, Protein expression and purification, vol. 70, no. 2, pp. 260-269, doi: 10.1016/j.pep.2009.09.012.

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Title Ligand-enhanced expression and in-cell assay of human peroxisome proliferator-activated receptor alpha ligand binding domain
Author(s) Velkov, Tony
Rimmer, Kieran A.
Headey, Stephen J.
Journal name Protein expression and purification
Volume number 70
Issue number 2
Start page 260
End page 269
Publisher Academic Press
Place of publication San Diego, Calif.
Publication date 2010-04
ISSN 1046-5928
1096-0279
Keyword(s) Peroxisome proliferator-activated receptor
alpha ligand binding domain
Nuclear hormone receptor
Ligand-enhanced protein expression
Summary A human peroxisome proliferator-activated receptor alpha ligand binding domain (PPARαLBD)-maltose binding protein fusion construct was expressed in Escherichia coli. A codon optimized DNA sequence encoding human PPARαLBD (aa196–468) was synthesized and ligated into the pDEST17 E. coli expression vector downstream of a MBP solubility fusion tag and an intermittent TEV protease cleavage site. Following auto-induction at 28 °C, PPARαLBD protein was purified to electrophoretic homogeneity by a nickel affinity chromatographic step, on-column TEV protease cleavage followed by Sephacryl S200 size exclusion chromatography. The recombinant protein displayed cross-reactivity with goat anti-(human PPARα) polyclonal antibody and was identified as human PPARα by trypic peptide mass finger-printing. The addition of a PPARα specific ligand (fenofibric acid, GW7647 or GW590735) to the growth media significantly stabilized the PPARαLBD structure and enhanced the expression of soluble protein. In-cell ligand binding was examined by monitoring the enhancement of PPARαLBD expression as a function of the concentration of ligand in the growth media. The efficient expression and in-cell assay of the reported PPARαLBD construct make it amenable to high through-put screening assays in drug discovery programs.
Language eng
DOI 10.1016/j.pep.2009.09.012
Field of Research 111599 Pharmacology and Pharmaceutical Sciences not elsewhere classified
Socio Economic Objective 970111 Expanding Knowledge in the Medical and Health Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2010, Elsevier
Persistent URL http://hdl.handle.net/10536/DRO/DU:30041037

Document type: Journal Article
Collection: School of Medicine
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