title: Functional reconstitution of human equilibrative nucleoside transporter-1 into styrene maleic acid co-polymer lipid particles.
abstract: The human equilibrative nucleoside transporter-1 (hENT1) is important for the entry of anti-cancer and anti-viral nucleoside analog therapeutics into the cell, and thus for their efficacy. Understanding of hENT1 structure-function relationship could assist with development of nucleoside analogs with better cellular uptake properties. However, structural and biophysical studies of hENT1 remain challenging as the hydrophobic nature of the protein leads to complete aggregation upon detergent-based membrane isolation. Here we report detergent-free reconstitution of the hENT1 transporter into styrene maleic acid co-polymer lipid particles (SMALPs) that form a native lipid disc. SMALP-purified hENT1, expressed in Sf9 insect cells binds a variety of ligands with a similar affinity as the protein in native membrane, and exhibits increased thermal stability compared to detergent-solubilized hENT1. hENT1-SMALPs purified using FLAG affinity M2 resin yielded ~0.4mg of active and homogenous protein per liter of culture as demonstrated by ligand binding, size-exclusion chromatography and SDS-PAGE analyses. Electrospray ionization mass spectrometry (ESI-MS) analysis showed that each hENT1 lipid disc contains 16 phosphatidylcholine (PC) and 2 phosphatidylethanolamine (PE) lipid molecules. Polyunsaturated lipids are specifically excluded from the hENT1 lipid discs, possibly reflecting a functional requirement for a dynamic lipid environment. Our work demonstrates that human nucleoside transporters can be extracted and purified without removal from their native lipid environment, opening up a wide range of possibilities for their biophysical and structural studies.
authors.affiliation: Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O.Box 3000, FI-90014, Oulu, Finland.
authors.forename: Shahid
authors.initials: S
authors.lastname: Rehan
authors.affiliation: Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Ville O
authors.initials: VO
authors.lastname: Paavilainen
authors.affiliation: Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O.Box 3000, FI-90014, Oulu, Finland. Electronic address: veli-pekka.jaakola@novartis.com.
authors.forename: Veli-Pekka
authors.initials: VP
authors.lastname: Jaakola
mesh_terms: D000818:Animals; D033721:Equilibrative Nucleoside Transporter 1 / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D008298:Maleates / Q000737:chemistry*; D008563:Membrane Lipids / Q000737:chemistry; D011137:Polystyrenes / Q000737:chemistry*; D055550:Protein Stability; D061987:Sf9 Cells; D012995:Solubility
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; D008298:Maleates; D008563:Membrane Lipids; D011137:Polystyrenes; C438649:SLC29A1 protein, human; C024074:styrene-maleic acid polymer
keywords: Lipid; Membrane proteins; Model membrane; Nucleoside transporter, SMALPs