A protocol for the combined sub-fractionation and delipidation of lipid binding proteins using hydrophobic interaction chromatography

https://doi.org/10.1016/j.jchromb.2008.04.011Get rights and content

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 γ 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.

Introduction

Intracellular long-chain fatty acids (FA) are key components in the synthesis of cellular membranes as well as being utilized as signaling molecules and for energy delivery [1]. The preservation of a proper balance between absorption, secretion, and storage of FA, is therefore, integral for cellular physiology. Increasingly prominent diseases such as obesity, cardiovascular diseases, type II diabetes, and atherosclerosis, to a large extent, all evolve from disorders of lipid metabolism [2]. Due to their poor solubility in water, transport of FA in vivo is via intracellular lipid binding proteins (iLBPs) [1], [3], [4]. The expression of genes involved in FA metabolism and glucose homeostasis is controlled by nuclear receptors, in particular a class termed the peroxisome proliferator-activated receptors (PPARs) [5], [6], [7], [8], [9]. PPARs are ligand-activated transcription factors that are activated by FA and eicosanoids [5], [6], [7], [8], [9].

Three isotypes of human PPAR, termed α, γ and δ, have been identified each with a specific tissue distribution [5], [6], [7], [8], [9]. PPARα and γ are the most studied isotypes. PPARα modulates FA metabolism and glucose homeostasis in the liver and skeletal muscle, whereas PPARγ modulates adipogenesis and adipocyte FA metabolism [5], [6], [7], [8], [9]. The physiological role of PPARδ is the least understood of the three human PPAR isotypes. However, not unlike the other two isotypes, PPARδ binds FA and eicosanoids, signifying a regulatory role in lipid metabolism [9]. Dysfunction of these aspects of biology leads to the aforementioned human diseases. Accordingly, PPARs are important targets for anti-dyslipidemic drugs [7], [8].

The intracellular trafficking mechanisms whereby lipid signaling molecules reach their nuclear receptor targets are not precisely known. Available evidence suggests the most likely candidates are a family of low molecular weight (12–15 kDa) iLBPs, collectively termed fatty acid binding proteins (FABPs) [1], [3], [4]. FABPs appear to act as intracellular shuttles for lipophilic ligands to the nucleus, where the ligand is released to PPARs, thereby effecting transcriptional regulation of metabolic enzymes and transporters that target the activating ligand [10], [11], [12], [13].

In light of the central regulatory role of PPARs and FABPs in lipid homeostasis, it follows that the development of novel therapeutic ligands with improved pharmacological profiles to target these iLBPs, has become an important research priority in the pharmaceutical industry [2], [7], [8]. However, the study of iLBP ligand binding affinities and the molecular interactions governing ligand selectivity are complicated due to the co-purification of high affinity endogenous lipids. Current protein delipidation methods are far from optimal, often operating under denaturing conditions. Research into the development of improved delipidation methodology that can be implemented on an industrial scale is severely lacking. Our interest in the characterization of PPAR and FABP ligand interactions lead us to test two commonly employed procedures for the delipidation of iLBPs. The organic solvent liquid–liquid extraction [14], [15] and lipidex 1000 [16], [17], [18] methods were compared to a novel hydrophobic interaction chromatography (HIC) procedure in order to develop new and improved delipidation methodology for aqueous lipid binding proteins. The presented HIC protocol combines delipidation with sub-fractionation of aqueous lipid binding proteins without denaturation of native protein structure.

Section snippets

Materials

Oleic acid, [1-14C], (54.6 mCi/mmol) was purchased from MP Biomedicals Australia (Seven Hills, N.S.W., Australia). 1-Anilino-8-naphthalene sulfonic acid (ANS), 8,8′-dianilino-5,5′-binaphthalene-1,1′-disulfonate (bisANS) and FA standards were obtained from Sigma–Aldrich (Sydney, NSW, Australia). Cis-parinaric acid was purchased from Invitrogen (Melbourne, VIC, Australia). Escherichia coli (E. coli) strain BL21 Codon Plus (DE3)-RIL was purchased from Stratagene (La Jolla, CA, USA). The following

Protein purification and characterization of protein-bound endogenous lipids

E. coli cells are routinely employed for the expression of mammalian proteins, owing to the simple and inexpensive culturing conditions, in addition to the high yields of recombinant protein produced. In our laboratory we employ E. coli BL21 cells for the expression of human and rat FABPs and PPAR LBDs and have established purification protocols [21]. The Ni2+-based IMAC purification of N-terminally [His]6 tagged human L-FABP and PPARγLBD, and HIC/anion exchange purification of I-BABP are given

Acknowledgements

We would like to thank Mrs. Anna Velkov for assistance with preparation of the manuscript and Dr Simon Egan for performing the mass spectrometry. We would also like to thank Associate Professor Christopher Porter and Dr Martin Scanlon for helpful discussions. T. Velkov is the recipient of a Peter Doherty Fellowship (384300) from the National Health and Medical Research Council, Australia.

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