Protocols and pitfalls in obtaining fatty acid-binding proteins for biophysical studies of ligand-protein and protein-protein interactions

Abstract

Adipocyte fatty acid-binding protein (AFABP: FABP4) is a member of the intracellular lipid-binding protein family that is thought to target long-chain fatty acids to nuclear receptors such as peroxisome proliferator-activated receptor gamma (PPARγ), which in turn plays roles in insulin resistance and obesity. A molecular understanding of AFABP function requires robust isolation of the protein in liganded and free forms as well as characterization of its oligomerization state(s) under physiological conditions. We report development of a protocol to optimize the production of members of this protein family in pure form, including removal of their bound lipids by mixing with hydrophobically functionalized hydroxypropyl dextran beads and validation by two-dimensional NMR spectroscopy. The formation of self-associated or covalently bonded protein dimers was evaluated critically using gel filtration chromatography, revealing conditions that promote or prevent formation of disulfide-linked homodimers. The resulting scheme provides a solid foundation for future investigations of AFABP interactions with key ligand and protein partners involved in lipid metabolism.

Associations between Fatty Acid-Binding Protein 4–A Proinflammatory Adipokine and Insulin Resistance, Gestational and Type 2 Diabetes Mellitus

“…Furthermore, FABP4 could be a treatment target in T2DM [45]. A small molecular ligand for FABP4 that blocks the binding of endogenous ligands may be developed into a drug for the treatment of T2DM [46].…”

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein (FABP1) and FABP1 Binding Alters Drug Metabolism

“…In the preliminary experiments, majority of the hFABP1 was observed bound with CPMs after nickel purification and before gel filtration and delipidation treatments (Figure 1A). Lipidex-5000 and butanol extraction have been reported to efficiently delipidate FABP1 (Velkov et al ., 2008; Wang et al ., 2017; Lai et al ., 2020). Various levels of CPMs were removed with individual treatments with Lipidex-5000, 1:1 (v/v) butanol and 1:3 (v/v) butanol based on the native MS analysis (Figure 1).…”

“…FABPs are promiscuous proteins that bind diverse ligands including native E. coli lipids and molecules present in expression and purification media (Velkov et al ., 2008; Wang et al ., 2017). Such ligands may be bound to recombinant purified FABPs as contaminating copurifying molecules (CPMs).…”

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism

“…This FABPs are promiscuous proteins that bind diverse ligands including native E. coli lipids and molecules present in expression and purification media (Velkov et al, 2008;Wang et al, 2017).…”

“…Lipidex-5000 and butanol extraction have been reported to efficiently delipidate FABP1 (Velkov et al, 2008;Wang et al, 2017;Lai et al, 2020). Various levels of CPMs were removed with individual treatments with Lipidex-5000, 1:1 (v/v) butanol and 1:3 (v/v) butanol based on the native MS analysis (Figure 1).…”