title: A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography.
abstract: Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor gamma ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.
authors.affiliation: Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville 3052 Victoria, Australia. Tony.Velkov@vcp.monash.edu.au
authors.forename: Tony
authors.initials: T
authors.lastname: Velkov
authors.forename: Maria L R
authors.initials: ML
authors.lastname: Lim
authors.forename: Benjamin
authors.initials: B
authors.lastname: Capuano
authors.forename: Richard
authors.initials: R
authors.lastname: Prankerd
mesh_terms: D002152:Calorimetry, Differential Scanning; D002853:Chromatography, Liquid / Q000379:methods*; D008024:Ligands; D008055:Lipids / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D008024:Ligands; D008055:Lipids; D011506:Proteins