Rehan, Shahid, Paavilainen, Ville O and Jaakola, Veli-Pekka (2017) Functional Reconstitution of Human Equilibrative Nucleoside Transporter-1 into Styrene Maleic Acid Co-polymer Lipid Particles. Biochimica et biophysica acta. ISSN 0006-3002

Abstract

The human equilibrative nucleoside transporter-1 (hENT1) is an 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 hENT1transporter 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.

Item Type:	Article
Date Deposited:	06 Mar 2017 00:45
Last Modified:	06 Mar 2017 00:45
URI:	https://oak.novartis.com/id/eprint/32377