Protein Expression and Purification 114 (2015) 99–107

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Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells Shahid Rehan, Veli-Pekka Jaakola ⇑ Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland

a r t i c l e

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Article history: Received 8 June 2015 Received and in revised form 3 July 2015 Accepted 7 July 2015 Available online 7 July 2015 Keywords: Equilibrative nucleoside transporter Anti-cancer Expression Purification Thermostability

a b s t r a c t 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 Kd of 1.2 ± 0.2 nM and Bmax 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 Kd 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. Ó 2015 Elsevier Inc. All rights reserved.

1. 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]. ⇑ Corresponding author at: Center for Proteomic Chemistry, Novartis Institute for Biomedical Research, Basel, Switzerland. E-mail address: veli-pekka.jaakola@oulu.fi (V.-P. Jaakola). http://dx.doi.org/10.1016/j.pep.2015.07.003 1046-5928/Ó 2015 Elsevier Inc. All rights reserved.

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].

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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 [13–15]. 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. 2. Materials and methods 2.1. Materials Phusion DNA polymerase and restriction enzymes were purchased from Thermo Scientific. Anti-FLAG M2 affinity resin, SLC29A1 anti-bodies and FLAG anti-bodies were from Sigma. Insect cell culture media was from Lonza. All detergents used were purchased from Anatrace. [3H]NBMPR with a specific activity of 8.43 Ci/mmol was purchased from Moravek Biochemicals. NBMPR, dilazep and dipyridamole were purchased from Tocris Bioscience. Filtermat B glass filters and MeltiLex B/HS scintillation wax was obtained from PerkinElmer.

tetracycline, 50 lg/ml kanamycine and 7.5 lg/ml gentamycine and incubated for 48 h at 37 °C. The transposase gene encoded in the helper plasmid of DH10Bac transfers the hENT1 gene into the Tn7 sites on the modified baculovirus genome, thereby disrupting the LacZ gene. Therefore, cells containing recombinant bacmid DNA forms white colonies, while the non-recombinant forms blue colonies. The white colonies were picked and re-streaked on a fresh plate for 48 h. The recombinant bacmid DNA was extracted using the phenol/chloroform method, PCR verified, and used to co-transfect insect cells using Invitrogen’s protocol. Recombinant baculovirus containing the hENT1 transporter gene was passed three times to generate high titer virus stock. GFP fusion was used to monitor the infection control and virus titer determination. The optimal expression level was achieved by screening for multiplicity of infection (MOI) and time of infection (TOI) using GFP fluorescence and the radioligand binding assay. 2.4. Saturation and competition binding experiments The dissociation constant Kd and total binding sites Bmax were measured by the saturation binding experiment as described previously [18]. Briefly, 1.5 lg of raw membrane obtained from Sf9 cells expressing hENT1 or LMNG solubilized membrane was diluted in transport buffer (10 mM Tris–HCl, pH 7.5, 0.1 mM MgCl2, 1 mM CaCl2) and incubated with graded concentration of [3H]NBMPR from 0.125 to 20 nM for 40 min at 22 °C. Non-specific binding was determined with 10 lM of dipyridamol. Unbound ligand was removed by washing the reaction mixture with ice-cold transport buffer on 0.4 lm 24 well Filtermat B glass filters (PerkinElmer) using a Brandel Harvester. Filtermats were dried and sealed with MeltiLex B/HS scintillation wax (PerkinElmer) and radioactivity was measured using a MicroBeta Trilux scintillation counter (PerkinElmer). The amount of [3H]NBMPR specifically bound to hENT1 was calculated as the difference between amount of [3H]NBMPR that bound in the presence and absence of 10 lM dipyridamole. Kd and Bmax were calculated by fitting the experimental data to a single-site specific binding equation using non-linear regression in Graphpad prism. For competition binding assays Sf9 crude membrane or the LMNG solubilized fraction was incubated with graded concentrations of either dilazep, dipyridamole or unlabelled NBMPR for 10 min before the addition of [3H]NBMPR. The inhibitory constant (Ki) values were calculated according to the method of Cheng and Prusoff [19]. 2.5. Detergent solubilization screening

2.2. Plasmid construction cDNA encoding full-length hENT1 (NCBI gene ID: 2030) was PCR amplified and through homologous recombination, inserted into a yeast expression vector pDDGFP1 (a kind gift from Prof So Iwata, Imperial College London) containing hENT1 gene and 8x His separated by a TEV protease cleavage site. This expression cassette was sub-cloned into a pFastBac1 vector using hENT1 specific primers containing sites for Sph I and Kpn I restriction enzymes. For large scale expression and purification hENT1 was cloned directly into the pFastBac1 vector with N-terminus FLAG tag and C-terminus His tag. All constructs were confirmed by DNA sequencing. 2.3. Recombinant baculovirus generation and expression in insect cells The pFastBac1 vector containing full length hENT1 gene was transformed into competent DH10Bac Escherichia coli cells and plated out onto LB plates containing 40 lg/ml Blue-gal, 10 lg/ml

Several commonly used detergents were tested for their ability to solubilize the hENT1 transporter, based on GFP fluorescence signal as described previously [20]. Detergent solubilized membrane protein-GFP fusion is run on SDS–PAGE and intensity of GFP signal is flowed by exciting at 488 nm and emission at 535 nm [21]. We selected 20 different detergents from all three classes i.e. ionic, nonionic and zwitterionic, including detergents most commonly used for membrane protein structural biology (Table 1). The final detergent concentration ranged between 5 and 200 critical micelle concentration (CMC). Typically, membrane isolated from insect cells was diluted to 2 mg/ml in solubilization buffer (50 mM HEPES, pH 7.4, 800 mM NaCl, 10% (v/v) glycerol) in a total reaction volume of 300 ll. Solubilization was done at 4 °C for 1 h followed by ultracentrifugation at 235,000g for 1 h. 10 ll of soluble fractions from each detergent were loaded on 10% SDS–PAGE for in-gel fluorescence analysis. GFP fluorescence was measured with 200 ll of the soluble fractions using Infinite 1000Pro plate reader

S. Rehan, V.-P. Jaakola / Protein Expression and Purification 114 (2015) 99–107 Table 1 List of detergents used in this study for extraction of hENT1. Detergents

CMC (%)

Concentration used in this study (% v/v)

DM DDM bOG LMNG OGNG C12E9 CYMAL-5 CYMAL-6 Fos-Choline-11 Fos-Choline-12 Big CHAP CHAPSO LAPAO DDAO TDAO

0.08 0.00870 0.53 0.001 0.05 0.0048 0.12 0.028 0.068 0.047 0.25 0.5 0.052 0.023 0.0075

1 1 2 1 1 1 2 1 1 1 2 3 1 1 1

(Tecan, Ex 485 nm/Em 535 nm). A BCA assay was used to determined total protein concentration in each detergent fraction and 5 lg of solubilized protein used for [3H]NBMPR binding activities as described above. 2.6. Thermal shift assay The stability of hENT1 in different detergents was monitored using a radioligand binding assay as described previously [22].

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Briefly, after solubilization 100 ll samples from each detergent were incubated at 4–80 °C in increments of 5 °C in a gradient thermocycler for 30 min. A Control sample was kept on ice. After heating, the samples were chilled on ice for 5 min and centrifuged at 235,000g for 30 min at 4 °C. [3H]NBMPR binding activities were measured using the method described above by normalizing the data with the untreated sample. The effects of different lipids on the stability of hENT1 were also determined. The stability of purified hENT1 transporter in LMNG was assessed using a thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl) phenyl] maleimide (CPM) assay as described previously [23]. Briefly, 50 lg of purified protein was mixed with 8 lg of CPM dye in a total reaction volume of 120 ll and assay was conducted at 25–80 °C. The melting temperature in each detergent was calculated by fitting the experimental data to a Boltzmann sigmoidal equation by non-linear regression using Graphpad prism. 2.7. Membrane preparation and purification of hENT1 One liter of Sf9 culture at 1.5  106 cells/ml was infected at M.O.I  2 with recombinant baculovirus containing hENT1. After 44 h, the virus infected insect cells were collected by centrifugation at 1000g for 5 min, resuspended in 2 insect cell lysis buffer (10 mM HEPES pH 7.4, 10 mM MgCl2, 200 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM PMSF) and lysed using a dounce homogenizer. The cell lysate was ultracentrifuged at 235,000g for 1 h to collect

Fig. 1. Snake plot model of hENT1. (a) Schematic illustration of TM topology of hENT1 transporter. (b) hENT1 construct used for initial expression trials. (c) Construct used for saturation and competition binding experiments, thermal shift assay and large scale purification.

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Fig. 2. Expression of hENT1 in Sf9 cells. (a) Fluorescence microscopy images of Sf9 cells expressing the hENT1-GFP fusion protein. FITC-fluorescence (left) and bright field BF (right) images taken with 20 objective lens with exposure time of 100 and 10 ms, respectively. Cell shows 100% infectivity after 48 h.p.i. Inset; Single cell images showing plasma membrane localization of recombinant hENT1 transporter. (b) [3H]NBMPR binding with intact Sf9 cells (1  106/ml) with different multiplicities of infection (M.O.I). (c) [3H]NBMPR binding with intact Sf9 cells after infection at different time points.

the membrane fraction. The membranes were washed at least 3 times with high salt buffer (50 mM HEPES, pH 7.4, 1 M NaCl, 40% glycerol), resuspended into membrane storage buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 40% glycerol) and stored at 70 °C until further analysis. The frozen membrane fraction was thawed on ice and solubilized for 2.5 h at 4 °C in solubilization buffer (50 mM HEPES, pH 7.4, 800 mM NaCl, 10% glycerol, 1 mM PMSF) containing 1% LMNG, 0.2% CHS at 2 mg/ml total protein concentration. The unsoluble fraction was separated by ultracentrifugation at 235,000g for 1 h. The soluble fraction was mixed with pre-equilibrated 1 ml of anti-FLAG M2 resin. Binding was done overnight at 4 °C with continuous mixing. Anti-FLAG M2 resin with bound protein was loaded onto a BioRad chromatography column and the flow through was collected by gravity flow. The resin was washed with 10–20 column volume of solubilization buffer containing 0.01% LMNG/0.002% CHS. The protein was eluted with the solubilization buffer containing 300 lg FLAG peptide and 0.005% LMNG/0.001% CHS. The purified protein was analyzed by SDS– PAGE for purity and by size-exclusion chromatography for homogeneity. 2.8. Mass spectrometry analysis The full length hENT1 transporter was purified, separated on SDS–PAGE and stained with Coomassie brilliant blue. A band corresponding to the expected size of hENT1 was excised, processed as described previously [24] and analyzed by LC/MS/MS. Database searches were performed by using the Mascot program. 2.9. Western blot analysis Western blotting was carried out by standard procedures. Purified transporter (0.5 lg) from the FLAG IMAC column was

separated on a 12% SDS–PAGE gel, and electroblotted to a carefully pretreated PVDF membrane in semi-dry conditions. The samples were immunoblotted with anti- FLAG and anti-SLC29A1 antibodies. 2.10. [3H]NBMPR binding with purified hENT1 transporter After elution from the anti-FLAG M2 column all purification fractions including 0.25 lg of purified hENT1 were incubated with 3.5 nM [3H]NBMPR in the presence and absence of 10 lM dipyridamole and processed as described above. Aliquots of equal amount of the purified protein were stored at 4, 20 and 70 °C and [3H]NBMPR binding was measured after one week. [3H]NBMPR saturation binding experiment with purified transporter was carried out as described above. 3. Results and discussion 3.1. Optimal expression conditions We initially used a GFP-based expression optimization strategy that has been previously used for membrane proteins [25]. Small scale expression of the hENT1-GFP fusion construct (Fig. 1b) in Sf9 cells showed the plasma membrane localization of the GFP fusion protein (Fig. 2a), which served as evidence of correct folding and localization of the recombinant transporter [25]. We tested the expression profile of the transporter and found that the optimal expression time is between 44 and 48 h after infecting Sf9 cells with recombinant virus with an M.O.I  2–5 (Fig. 2b). A longer expression time resulted in a significant increase in proteolytic degradation (data not presented) and relatively modest further increase in functional expression as indicated by the radioligand

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Fig. 3. Saturation and competition binding experiment of hENT1. (a) Equilibrium binding of [3H]NBMPR (0.12–20 nM) to Sf9 membranes expressing hENT1 transporter. The results are presented as the amount of specifically bound (i.e. Total binding – non-specific binding) [3H]NBMPR as a function of free [3H]NBMPR. Each point is the average of at least three measurements. Three experiments gave similar results yielding a Kd of 1.2 ± 0.2 nM and Bmax of 110 ± 5 pmol/mg of protein. Inset to a: Scatchard plot of specific binding of [3H]NBMPR to Sf9 membranes. (b) Displacement of [3H]NBMPR binding to hENT1 by different inhibitors. Sf9 membrane expressing hENT1 incubated with 3.5 nM [3H]NBMPR alone or together with the graded concentration (0.01 pM–100 lM) of either unlabelled NBMPR, dilazep or dipyridamole. Results are shown as the percentages of [3H]NBMPR bound as a function of the logarithm of the concentration of unlabeled NBMPR, dilazep and dipyridamole. The amount of [3H]NBMPR that bound in the absence of inhibitors was taken as 100% binding. Each point is the average of at least three measurements. Three experiments gave similar results yielding Ki values for NBMPR, dilazep and dipyridamole of 2.5 ± 0.1, 2.7 ± 0.1 and 15.7 ± 0.1 nM, respectively. (c) Equilibrium binding of [3H]NBMPR (0.12–20 nM) to LMNG solubilized Sf9 membranes expressing hENT1 transporter. Each point is the average of at least three measurements. Two experiments gave similar results yielding a Kd of 6.9 ± 2.4 nM and a Bmax of 19.6 ± 2.6 pmol/ mg of protein. Inset to c: Scatchard plot of specific binding of [3H]NBMPR to LMNG solubilized hENT1 transporter. (d) Displacement of [3H]NBMPR binding to LMNG solubilized hENT1 performed as described above. Each point is the average of at least three measurements. Two experiments gave similar results yielding Ki values for NBMPR, dilazep and dipyridamole of 25 ± 1, 37 ± 1 and 303 ± 1 nM, respectively.

binding assay (Fig. 2c). We then, used a construct with an N-terminal FLAG-tag and a C-terminal His-tag (Fig. 1c) for saturation and competition binding, thermal shift assay and large scale purification. The hENT1 transporter has been expressed mainly in mammalian cells and Xenopus laevis oocytes for functional studies [16]. Yeast expression of hENT1 was mainly targeted for functional studies from native membrane or proteoliposomes [18]. In this paper, we present a protocol for overexpression and purification of hENT1 from Sf9 cells in a fully functional state. 3.2. [3H]NBMPR binding activities We demonstrated that the heterologous expression of hENT1 in Sf9 cells yielded saturable binding of [3H]NBMPR with the Kd of 1.2 ± 0.2 nM and number of active binding sites Bmax of 110 ± 5 pmol/mg of protein (Fig. 3a). The affinity of [3H]NBMPR for the recombinant transporter observed in this study was similar to those found in native es transporters of erythrocytes and cultured cells [26], suggesting successful functional expression in insect cells. In the competition binding experiment of [3H]NBMPR with Sf9 membranes, we obtained Ki values of 2.5 ± 0.1, 2.7 ± 0.1 and 15.7 ± 0.1 nM, for unlabeled NBMPR, dilazep and dipyridamole, respectively (Fig. 3b). However, decrease in the binding affinity of [3H]NBMPR and other inhibitors to hENT1 was observed when the protein was extracted into LMNG detergent as the Kd values increased to 6.9 ± 2.6 nM (Fig. 3c). This was

not surprising since even the mildest detergents cannot mimic the native membrane environment due to delipidation and potential structural re-arrangements during solubilization. The slight alteration in Kd of [3H]NBMPR between the membrane and solubilized fractions may also be attributed to the non-specific interaction between two amphipathic molecules i.e., detergent and inhibitor [27]. Unlabeled NBMPR, dilazep and dipyridamole exhibit competitive inhibition of [3H]NBMPR binding to hENT1 and other es transporters [28]. This was demonstrated in a competition binding experiment of [3H]NBMPR with Sf9 membranes and LMNG solubilized hENT1, which yielded a rank order of binding (Fig. 3d), that corresponds with literature data. Nevertheless, maintaining the rank order and high affinity binding suggest that LMNG preparations have retained the nucleoside recognition site (in this case the NBMPR binding site) of the transporter in nearly a wild-type like functional state. 3.3. Detergent screen For structural and other biophysical studies membrane proteins are required to be isolated and incorporated into detergent micelles. It is crucial to find a suitable detergent that ensures the homogeneity, stability and activity of membrane protein. Since such experiments for the hENT1 transporter remain challenging [29], we have taken this task to screen several different detergents. In our study, most of detergents were able to extract hENT1 efficiently except CYMAL-1 (Fig. 4a). We used Fos-Choline series

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Fig. 4. Systematic detergent screening of hENT1. (a) In-gel fluorescence SDS–PAGE image of the detergent solubilization screen of the hENT1-GFP fusion protein. (b) [3H]NBMPR binding activities in each detergent measured as described in Section 2. (c) eGFP fluorescence measurement of solubilized fractions from each detergent used to calculate solubilization efficiencies using fluorescence of an equal amount of unsolubilized membrane as 100%. All experiments were repeated at least twice.

as ‘‘total’’ extractors as their solubilization efficiencies are highest (Fig. 4a and c) but [3H]NBMPR binding activities in these detergents were similar to those of the inactive transporter (Fig. 4b). These results were not surprising since the zwitterionic nature of these detergents is known to inactivate many membrane proteins. Another trend was seen in the CYMAL series; detergents with an increase in carbon chain length increased the solubilization efficiencies. A similar trend was seen in n-tetradecyl-N,N-dimethylamine-N-oxide (TDAO) and n-decyl-N, N-dimethylamine-N-oxide (DDAO) detergents. On the other hand, Big CHAP solubilized reaction turned into cloudy precipitate upon longer incubation and was therefore not included in further screening despite relatively high [3H]NBMPR binding activities. Detergents such as DM, DDM, LMNG, DMNG, CYMAL-6 with moderate solubilization efficiencies and relatively higher [3H]NBMPR binding activities were used for further screening. Current study exploited the use of GFP-based detergent solubilization screening complemented with in-gel fluorescence. Our method is inexpensive, fast and simple compared to typical FSEC-based detergent screening which require large quantities of detergents. Moreover, this method is relatively less time consuming compared to the western blotting based screening and can be adapted to multiple targets at the same time, eliminating the need of protein specific antibody. These results correspond well with the dot blot data of similar detergent screen performed with non-GFP fusion construct of the hENT1 transporter (Supplementary Fig. 1). 3.4. Melting curves of hENT1 The thermal stability of hENT1 protein (non-GFP fusion, Fig. 1c) in different detergents was measured according to the method described previously [22]. The function of the transporter was

probed by a thermostability assay based on radioligand binding which allows heat perturbation of small samples solubilized in different detergents. The affinity of hENT1 for the classical inhibitor NBMPR at different incubation temperatures can be fitted to a sigmoid curve describing the thermotrophic transition from the folded to the unfolded form of the transporter. From the inflection point in a sigmoid curve, an apparent Tm can be calculated, signifying the temperature at which 50% of the ligand binding is lost. The melting temperature (Tm) of the hENT1 transporter in native Sf9 membranes is 47 ± 1 °C but it decreased to 30 °C in DDM (data not shown) and other maltoside detergents (Fig. 5a). Most of the detergents showed similar melting curves with the exception of LMNG which demonstrate remarkable stabilizing effects on hENT1 with a Tm of 43 °C. Cholesteryl hemisuccinate (CHS) and asolectin have further increased the thermal stability of the LMNG solubilized transporter and increased the Tm  4 degree above the calculated Tm in Sf9 membranes (Fig. 5b). However, we are not sure whether this stabilizing effect is caused by the directed interaction between lipid and the transporter or via changes in micelle structure [21]. Indeed, this was surprising since the stability of membrane proteins in native membranes is expected to be higher than in a detergent solubilized form. It may also imply that local lipid concentrations in Sf9 membrane may not be sufficient for optimal stability of the transporter. For example, Sf9 cells are known to have low cholesterol levels. In our opinion this unexpected Tm difference could also be due to the fact that Sf9 membranes are not the native source for this protein and the Tm in mammalian cell membrane would be much higher than in Sf9 membranes. CPM dye specifically binds to the cysteine amino acid which often resides in the hydrophobic folds of the protein. Unbound CPM dye is non-fluorescent but becomes fluorescent upon binding

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Fig. 5. Thermostability analysis of hENT1 in different detergent/lipid environments. (a) Radioligand binding-based thermal shift assay of hENT1 (Non-GFP fusion) in various detergents. The protein was solubilized and heated in a thermocycler for 30 min at 4–80 °C and [3H]NBMPR binding activities were measured as described in Section 2. All experiments were repeated at least three times. Each point is the average of at least three measurements. (b) hENT1 thermal denaturation curve in the presence of different lipids. (c) Apparent Tm of hENT1 in different lipids and detergents compositions. (d) CPM assay of purified hENT1 in LMNG/CHS as described in Section 2.

with reactive cysteines, thus can be used as sensor to probe the integrity of membrane protein structure. The Tm of the purified hENT1 transporter was 70 ± 1 °C when determined using a CPM assay (Fig. 5d), which is much higher than the Tm calculated with radioligand binding. This is not surprising since the CPM assay uses the binding of dye to the native cysteines embedded in the protein interior as an indicator for the overall integrity of the folded state. The configuration of cysteines could possibly be retained at temperatures above those at which the transporter loses its ligand binding ability. Recently, it has been shown that CPM dye actually does not even require cysteine residues and can bind to large hydrophobic patches of the protein upon unfolding [23]. The LMNG and DM solubilized hENT1 transporter showed variable stability at 4 °C (Supplementary Fig. 2). DM solubilized transporter lost more than 50% of its [3H]NBMPR binding activities during the first 48 h of incubation while LMNG preparations retained nearly 80% of [3H]NBMPR binding after 48 h although this was reduced to 20% after 96 h. The observed gradual decrease in total binding with no major change in non-specific binding signified that this loss was specific for [3H]NBMPR binding sites and not for the overall sample due to prolonged incubation at 4 °C. In our experiments, DM, ßOG and CYMAL series detergents showed comparable [3H]NBMPR binding and solubilization efficiencies to LMNG but lack the stabilizing effect and therefore excluded from major purification attempts. 3.5. Characterization of purified hENT1 transporter Non-GFP fusion construct of hENT1 was expressed in 1 L of Sf9 cells and used for large scale purification. We initially used Ni–NTA

Table 2 Yield of hENT1 in each step of purification from Sf9 cells.

a b

Fraction

Yield (mg)

Purity (%)

Sf9 membranea LMNG-solubilized a FLAG purificationb

5.2 1.8 0.5

– – >90

Total protein estimated by BCA assay. hENT1 protein was estimated using nano-drop spectrometer (280 nm).

affinity resin but it failed to give sufficient purity of the transporter. After screening most of the commercial resins, we found that anti-FLAG M2 resin gives sufficient purity when analyzed on SDS–PAGE. We also found that washing the membrane fraction several times before solubilization reduced the non-specific binding of impurities and increased the final yield of hENT1 to 0.5 mg/L of culture (Table 2). We were able to purify functional hENT1 transporter as shown by [3H]NBMPR binding activities of different purification fractions and saturation binding experiment with eluates, yielding a Kd of 4.3 ± 0.7 nM (Fig. 6d). Kd value of purified protein was close to the value determine with Sf9 membrane, supporting argument of properly folded protein. Purified glycosylated hENT1 separated on 12% SDS–PAGE had an apparent Mr of 45–50 kDa and was detected with both anti-FLAG and anti-SLC29A1 antibodies (inset to Fig. 6b). Theoretical size of unglycosylated hENT1 is 52.2 kDa, however, slight shift on SDS–PAGE is expected as many membrane proteins show anomalous behavior during electrophoresis [30]. The hENT1-LMNG/CHS/protein-deter gent micelle complex eluted near 140 kDa on size exclusion chromatography which suggests monomeric transporter per micelle composition (Fig. 6b). Mass peptide fingerprint spectroscopy of

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Fig. 6. Characterization of purified hENT1 transporter. (a) [3H]NBMPR binding activities of different fractions of hENT1 purification. 1, 2, 3 are fraction from 1st, 2nd and 3rd elution from FLAG M2 column, respectively. (b) Size exclusion chromatography analysis of purified hENT1 on a Superdex G 200 10/300 GL column with a flow rate of 0.3 ml/ min. Relative size of protein-detergent complex was estimated using elution profiles of protein standards from UV, light scattering and RI detectors. Inset to b; lane 1 purified protein (2.5 lg) loaded on 12% SDS–PAGE (monomer ‘‘one star’’ and SDS-resistant aggregate ‘‘two stars’’) and visualized with simple blue staining. Lane 2 and lane M are BSA standard (20 lg) and protein marker, respectively. Lane 3 and 4 are western blot analyses with anti-SLC29A1 and anti-FLAG, antibodies, respectively. (c) The effect of storage temperatures on purified protein determined by radioligand binding assay as described in main text. [3H]NBMPR binding activities of an equal amount of protein immediately after purification was considered as control. (d) Saturation binding experiment of the purified hENT1 transporter with [3H]NBMPR performed as described above for Sf9 membrane and LMNG solubilized protein, yielding a Kd of 4.3 ± 0.7 nM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the excised band near 50 kDa on SDS–PAGE was identified as hENT1 (with 13% sequence coverage). However, a minor band near 120 kDa on SDS–PAGE and 600 kDa on SEC analysis was designated as SDS-resistant aggregate since it was also detected by in-gel fluorescence analysis of hENT1-eGFP fusion protein (Supplementary Fig. 3) and showed no [3H]NBMPR binding after fractionated on gel filtration. The effect of storage conditions on the nucleoside binding site of purified hENT1 was assessed by storing the samples at 4, 20 and 70 °C. [3H]NBMPR biding activities measured after one week showed that the transporter retained almost 90% of the ligand binding activity when stored at 70 °C (Fig. 6c) with a slight decreased at 20 °C. Nearly 50% of the activity was lost if the transporter was stored at 4 °C. The difference between the thermal stability of LMNG solubilized and purified protein at 4 °C may be due to detergent-mediated protein aggregation in solubilized fractions.

4. Conclusion Our biochemical understanding of mammalian ENTs is very limited, despite their pharmacological relevance in several disease conditions. Structural studies of hENT1 are hampered by its low expression levels and instability during solubilization and purification in detergents. In this paper, we optimized overexpression, solubilization and purification steps using GFP-fusion and the

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