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

Tony Velkov, Kieran Rimmer, Stephen Headey

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7 Citations (Scopus)


A human peroxisome proliferator-activated receptor alpha ligand binding domain (PPARaLBD)-maltose binding protein fusion construct was expressed in Escherichia coli. A codon optimized DNA sequence encoding human PPARaLBD (aa196a??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, PPARaLBD 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 PPARa) polyclonal antibody and was identified as human PPARa by trypic peptide mass finger-printing. The addition of a PPARa specific ligand (fenofibric acid, GW7647 or GW590735) to the growth media significantly stabilized the PPARaLBD structure and enhanced the expression of soluble protein. In-cell ligand binding was examined by monitoring the enhancement of PPARaLBD expression as a function of the concentration of ligand in the growth media. The efficient expression and in-cell assay of the reported PPARaLBD construct make it amenable to high through-put screening assays in drug discovery programs.
Original languageEnglish
Pages (from-to)260 - 269
Number of pages10
JournalProtein Expression and Purification
Issue number2
Publication statusPublished - 2010

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