Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells

Highlights

• We cloned, expressed and purified human equilibrative nucleoside transporter-1 from Sf9 insect cells.

• We characterized the recombinant transporter by saturation and competition binding experiments.

• We determined the thermal stability of transporter in different detergent and lipid environment.

• We purified hENT1 in lauryl maltose neopentyl glycol detergent with a final yield of 0.5 mg/L.

• We showed that purified protein is stable, homogenous, active and ready for further biophysical and structural studies.

Abstract

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure–function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a K_d of 1.2 ± 0.2 nM and B_max of 110 ± 5 pmol/mg for [³H]nitrobenzylmercaptopurine ribonucleoside ([³H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a K_d of 4.3 ± 0.7 nM. The yield of hENT1 from Sf9 cells was ∼0.5 mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies.

Introduction

The biological membrane makes an ideal barrier across cellular compartments that allow some small molecules such as CO₂ and other gasses to pass through but is impermeable for other larger and nonlipophilic molecules. Therefore, most molecules require a specialized membrane transport system to facilitate their entrance into and exit from eukaryotic and prokaryotic cells. The role of transporter proteins in absorption, distribution and elimination of important drugs is well established [1], [2]. In the membrane transport system, nucleoside transporters (NTs) constitute a family of integral membrane proteins of the solute carrier (SLC) family [3]. In mammalian cells, two major families of nucleoside transporters exist; the SLC28 family of concentrative sodium dependent transporters (CNTs) and the SLC29 family of equilibrative sodium independent transporters (ENTs) [1]. Members of both families transport natural nucleosides as well as nucleoside analogs used to treat various types of cancers, HIV and many other viral diseases [4], [5].

The SLC29 family consists of four members (ENT1-4) of equilibrative nucleoside transporters. These proteins are important in the uptake of natural nucleosides which are a precursor to DNA/RNA synthesis. In particular, hENT1 plays an important role in regulation of the anti-inflammatory molecule adenosine which acts on cell surface adenosine receptors and mediate several physiological processes including vasodilation, cardioprotection, hormones and neurotransmitter release, platelet aggregation and lypolysis [6]. hENT1 is a 456 amino acids protein and predicted to have 11 alpha helical transmembrane (TM) domains with an apparent molecular weight of 52 kDa [7]. hENT1 is the major plasma membrane nucleoside transporter present in almost all tissue types although its relative abundance varies [8]. hENT1 deficient cells demonstrate resistance to several anticancer nucleosides and its abundance may determine the response to nucleoside drugs in some cancers. The uptake of nucleosides and nucleoside analogs by hENT1 is inhibited by exposure to nano-molar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), a specific and high affinity inhibitor of hENT1. Initially NBMPR was used to differentiate nucleoside transporters as equilibrative-sensitive (es) and equilibrative-insensitive (ei) transporters [9].

High resolution structural information is indispensable for the development of structure based drug design [10], [11]. Structural studies require well-diffracting crystals obtained from purified proteins in mg quantities. Structural studies of membrane proteins are hampered by their intrinsic hydrophobic nature, and require the choice of an appropriate expression host, promoter system, purification tags, post translational modifications and the use of a suitable detergent to maintain the protein in its native conformation after isolation from the membranes [12]. Despite these challenges several membrane proteins including G-protein coupled receptors, ion-channels and transporters have been successfully expressed, purified and crystallized [13], [14], [15]. ENTs have so far been expressed primarily in mammalian cells and Xenopus laevis oocytes for cell biology experiments [16].

To date, the structure of both ENTs and CNTs are unknown with the exception of the functionally distinct Vibrio cholera CNT [17]. Despite extensive biochemical and pharmacological studies, little is known about the structure–function relationship of hENT1. Structural information such as the nucleoside binding mechanism, conformational changes during binding and transportation of nucleoside drugs are elusive. Such information would be helpful in designing novel drugs with better efficacy, which could greatly improve current chemotherapies. The challenges related to the structural studies of ENTs are multiple: low expression level, intrinsic transporter structural flexibility, solubilization from the membrane and stability in detergent after solubilization. To overcome these problems we have developed a protocol for overexpression and functional production of hENT1 in Sf9 insect cells. Lauryl maltose neopentyl glycol (LMNG) detergent purified hENT1 protein is stable, active, and homogenous and is appropriate for further biophysical and structural studies.