Ligand-enhanced expression and in-cell assay of human peroxisome proliferator-activated receptor alpha ligand binding domain.
A human peroxisome proliferator-activated receptor alpha ligand binding domain (PPAR alpha LBD)-maltose binding protein fusion construct was expressed in Escherichia coli. A codon optimized DNA sequence encoding human PPAR alpha LBD (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 degrees C, PPAR alpha LBD 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 PPAR alpha) polyclonal antibody and was identified as human PPAR alpha by trypic peptide mass finger-printing. The addition of a PPAR alpha specific ligand (fenofibric acid, GW7647 or GW590735) to the growth media significantly stabilized the PPAR alpha LBD structure and enhanced the expression of soluble protein. In-cell ligand binding was examined by monitoring the enhancement of PPAR alpha LBD expression as a function of the concentration of ligand in the growth media. The efficient expression and in-cell assay of the reported PPAR alpha LBD construct make it amenable to high through-put screening assays in drug discovery programs.
School of Medicine, Deakin University, Pigdons Road, Geelong, 3217 Victoria, Australia. Velkov@deakin.edu.au
Tony
T
Velkov
Kieran A
KA
Rimmer
Stephen J
SJ
Headey
D002087:Butyrates / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D011345:Fenofibrate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D058619:Maltose-Binding Proteins; D056913:Mediator Complex Subunit 1 / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D047493:PPAR alpha / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D033902:Periplasmic Binding Proteins / Q000235:genetics; D010671:Phenylurea Compounds / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology
C518308:2-methyl-2-(4-(((4-methyl-2-(4-trifluoromethylphenyl)-thiazol-5-ylcarbonyl)amino)methyl)phenoxy)propionic acid; D002087:Butyrates; D005231:Fatty Acids, Unsaturated; C453899:GW 7647; D008024:Ligands; D058619:Maltose-Binding Proteins; D056913:Mediator Complex Subunit 1; D047493:PPAR alpha; D033902:Periplasmic Binding Proteins; D010671:Phenylurea Compounds; D011422:Propionates; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; C006012:fenofibric acid; C013641:parinaric acid; D011345:Fenofibrate