A2AR expression, purification, and nanodisc reconstitution

Expression, purification, and reconstitution of the adenosine A_2A receptor in small phospholipid nanodiscs with ¹⁹F-labeling

Shuya Kate Huang¹ and R. Scott Prosser^1,2

¹ Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.

² Department of Biochemistry, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada.

Introduction

The adenosine A_2A receptor (A_2AR) is a widely-expressed eukaryotic cell-surface receptor activated by the endogenous ligand adenosine. It is a member of the class-A G protein-coupled receptor (GPCR) and a target for the treatment of inflammation, cancer, and various cardiovascular, respiratory, and central nervous system disorders (Effendi et al., 2020; Guerrero, 2018; Ruiz et al., 2014; Yu et al., 2020; Zheng et al., 2019). The following protocol describes the recombinant production of the human A_2AR for biophysical studies, where the receptor is expressed and purified in milligram quantities, reconstituted in small phospholipid nanodiscs, and labeled with a fluorinated tag for the purpose of fluorine (¹⁹F) nuclear magnetic resonance (NMR) experiments. The procedures for construct design and generation of the yeast strain have been documented in detail elsewhere (Ye et al., 2018, 2016). Briefly, Pichia pastoris (P. pastoris) SMD 1163 (Δhis4 Δpep4 Δprb1) cells were transformed with linearized pPIC9K plasmids carrying the human A_2AR gene featuring A_2AR residues 2-317 with a FLAG tag at the N-terminus and a His₁₀ tag at the C-terminus. The construct carries a single cysteine mutation, V229C, for ¹⁹F-labeling on transmembrane helix 6. The current protocol focuses on the expression, purification, and reconstitution of this receptor into small nanodiscs using the MSPΔH5 membrane scaffold protein. Some of these procedures have been described in our previously published work (Huang et al., 2022, 2021; Ye et al., 2016). For completeness, we also provide a protocol for the expression and purification of MSPΔH5, which was originally developed by (Hagn et al., 2013).

Materials and Reagents

Escherichia. coli BL21(DE3) cells (Invitrogen, catalog number: C600003)

pET28a-MSP1D1delltaH5 plasmid (Addgene, plasmid #71714)

P. pastoris SMD 1163 cells carrying high copy number of the gene encoding A_2AR-V229C (Ye et al., 2018, 2016)

Ampicillin

G418

Kanamycin

Isopropyl-β-D-thiogalactopyranoside (IPTG)

Antifoam A

Methanol

Materials for SDS-PAGE

2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide (BTFMA) (Apollo Scientific, catalog number:  PC8478)

Xanthine amine congener (XAC) (Santa Cruz Biotechnology, catalog number: sc-255717A)

Lauryl Maltose Neopentyl Glycol (LMNG) (Anatrace, catalog number NG310)

Cholesteryl hemisuccinate (CHS) (Sigma-Aldrich, catalog number C6512)

1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalog number: 850457C)

1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) (Avanti Polar Lipids, catalog number 840457C)

Bio-Beads SM-2 Resin (Bio-Rad, catalog number: 1523920)

Immobilized metal affinity resin (e.g., cOmplete His-Tag Purification Resin from Millipore Sigma/Roche)

TALON metal affinity resin     (Takara, catalog number: 635503)

Affi-Gel 10 (Bio-Rad, catalog number: 1536099)

Liquid nitrogen for flash-freezing

Luria Broth (LB) media

YPD plates (see recipes)

YPD media (see recipes)

BMGY media (see recipes)

BMMY media (see recipes)

MSP Lysis buffer (see Recipes)

MSP wash buffer A (see Recipes)

MSP wash buffer B (see Recipes)

MSP elution buffer (see Recipes)

TEV cleavage buffer (see Recipes)

Cholate storage buffer (see Recipes)

Cell washing buffer (see Recipes)

A_2AR lysis buffer (see Recipes)

Membrane solubilization buffer (see Recipes)

Labeling buffer (see Recipes)

LMNG storage buffer (see Recipes)

XAC elution buffer (see Recipes)

HNE buffer (see Recipes)

HNE buffer with 100 mM cholate (see Recipes)

Nanodisc storage buffer (see Recipes)

Equipment

Incubator at 30 °C and 37 °C

Temperature-controlled shaking incubator

FPLC instruments with capacity for column cooling

Cooled centrifuges

Cooled ultracentrifuge

Sonicator cell disruptor (e.g., Branson Ultrasonics Sonifier™ SFX150 Cell Disruptor)

Microfluidizer (e.g., LM 20, ATS Scientific)

Nanodrop spectrophotometer (or equivalent UV-vis spectrophotometer)

-20 °C and -80 °C freezers

Autoclave

Homogenizer

Magnetic stir plate

Roller mixer or orbital mixer for tubes

Baffled culture flasks

Benchtop laboratory balances

HiLoad 16/600 Superdex 200 pg size exclusion column (Cytiva, catalog number: GE28-9893-35)

Gravity columns

10,000 Da, 30,000 Da, and 50,000 Da MWCO spin-concentrators

0.2 µm PES or PVDF syringe filters (for filtering aqueous solutions)

0.2 µm nylon bottle top vacuum filters (for filtering DMSO and methanol)

20 mL scintillation vials

Centrifuge bottles for harvesting cells

Conical tubes

Ultracentrifuge tubes

Procedure

A. Expression and purification of MSPΔH5

1. Inoculate 20 mL of LB medium containing 50 µg/mL kanamycin with a single colony of E. coli BL21 (DE3) cells harboring the MSPΔH5 expression plasmid (originally developed by (Hagn et al., 2013) and available from Addgene (pET28a-MSP1D1delltaH5, plasmid #71714)). Grow the starter culture overnight at 37 °C with shaking.
2. Transfer the starter culture to 1 L of LB medium containing 50 µg/mL kanamycin. Grow the cells at 37 °C with shaking until the optical density at 600 nm (OD₆₀₀) reaches 0.6.
3. Induce the cells for 4 h at 37 °C with isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration 0.5 mM.
4. Harvest the cells by centrifugation at 6,000 g for 10 min. The cell pellet can be stored at -20 °C for up to 4 months.
5. Resuspend the cells in 40 mL of ice-cold MSP Lysis buffer.
6. Keeping the cells on ice, lyse the cells by sonication for 3 min with pulses of 10 s on and 10 s off and an amplitude of 50%.
7. Clear the lysate by centrifugation at 20,000 g for 30 min.
8. Load the supernatant into an immobilized metal affinity column. Wash the column with 5 bed-volumes of MSP wash buffer A, followed by 5 bed-volumes of MSP wash buffer B. Elute the protein with 5 bed-volumes of MSP elution buffer.
9. Buffer-exchange the eluted proteins to TEV cleavage buffer using a 10,000 Da MWCO spin-concentrator, until the imidazole concentration is below 5 mM. Bring the volume up to 15-20 mL with TEV cleavage buffer.
10. Add TEV protease to the His₆-TEV-MSPΔH5 sample at a final protease concentration of 30 µg/mL. Sterile-filter the sample through a 0.2 µm PES or PVDF syringe-filter into a sterile conical tube and incubate the reaction at 4 °C for 2-3 days for complete cleavage.
11. Load the sample on an immobilized metal affinity column to remove the TEV protease and the cleaved His₆ tag. Collect the flow-through and run an SDS-PAGE gel to check that this fraction contains only cleaved MSPΔH5. Presence of His-tagged MSPΔH5 will interfere with downstream separation of empty and receptor-embedded nanodiscs.
12. Buffer exchange the cleaved MSPΔH5 to cholate storage buffer using a 10,000 Da MWCO spin-concentrator and concentrate the sample to 0.5-2 mM for storage.
13. Determine protein concentration using a nanodrop spectrophotometer, using the extinction coefficient of MSPΔH5 at 280 nm and blanking with cholate storage buffer. Aliquot the protein and store at -20 °C for up to 12 months.

B. A_2AR expression

1. Use a sterile toothpick or inoculating loop to streak a YPD agar plate containing 0.5 mg/mL G418 from a glycerol stock of P. pastoris SMD 1163 (Δhis4 Δpep4 Δprb1) containing high copy number of the gene encoding A_2AR-V229C (Ye et al., 2018, 2016).
2. Incubate the plate at 30 °C until colonies are at a size of ~2 mm in diameter. This typically takes ~4-5 days. Leave a tray of water in the incubator to prevent the plate from drying out during this time.
3. For each liter of the final culture, prepare a small starter culture by inoculating a single colony from the YPD plate in 10 mL of YPD medium containing 0.2 mg/mL G418. Grow the cultures at 30 °C with shaking at 250 rpm for 12-24 h. Use loosely-capped sterile conical tubes or culture tubes.
4. Inoculate each of the 10 mL starter cultures into baffled flasks containing 200 mL of BMGY medium supplemented with 50 µg/mL kanamycin. Grow the cultures at 30 °C with shaking at 250 rpm until an OD₆₀₀ of 2-6. This typically takes 24 h. Switching the antibiotic from G418 to kanamycin allows faster yeast growth at this stage.
5. Inoculate each of the 200 mL BMGY culture into 1 L of BMMY medium (without methanol) in baffled flasks containing 50 µg/mL kanamycin or 100 µg/mL ampicillin. Incubate the cultures at 18-20 °C for 4 hours with shaking at 250 rpm. This allows the cells to stabilize at the lower temperature while using the remaining glycerol.
6. Induce the cells by adding sterile methanol to a final concentration of 0.5% (e.g., 6 mL methanol per 1.2 L of total culture volume). Add an additional 6-8 mL of methanol to the 1.2 L culture every 12-16 h, for a total induction time of 60-72 h at 18-20 °C.
7. Harvest the cells by centrifugation at 4,000 g for 20 min. Resuspend the cell pellet in cell washing buffer, and centrifuge again at 4,000 g for 20 min to collect the cell pellet. Flash-freeze the cell pellet in liquid nitrogen and store the cells at -80 °C for up to 6 months.

C. A_2AR purification and nanodisc reconstitution

1. Resuspend the cells in A_2AR lysis buffer. 4 mL of buffer is typically used per 1 g of wet cell mass. Less volume may result in blockage of the microfluidizer, while a larger volume may prolong the subsequent centrifugation steps.
2. Lyse the resuspended cells using a microfluidizer, passing twice at 20,000 psi. Always keep the extruder and the lysate on ice to prevent overheating of the lysate. Note: excessive lysis could result in smaller membrane pieces that take longer to pellet during ultracentrifugation.
3. Centrifuge the lysate at 8,000 g for 30 min at 4°C to remove intact cells and large cell debris.
4. Centrifuge the supernatant at 100,000 g for 60 min at 4°C to isolate the membrane fraction.
5. Resuspend the membrane pellet in membrane solubilization buffer, using a homogenizer or by pipetting up and down using a P1000 or a P5000 pipettor. 5 mL of buffer is typically used per 1 mL of membrane.
6. Gently stir the membrane resuspension at 4 °C for 24-48 h to allow adequate solubilization and extraction of the membrane proteins.
7. Centrifuge the membrane resuspension at 9,000 g for 1 h to remove large insoluble debris (note: the supernatant will remain cloudy). Incubate the supernatant with TALON metal affinity resin (~1 mL bed volume per 10 mL of membrane resuspension) washed with membrane solubilization buffer and gently stir the mixture at 4°C overnight.
8. Perform the next three steps if ¹⁹F-labeling is required.
9. Load the A_2AR-bound TALON resin in a gravity column and wash the resin with 5 column-volumes of labeling buffer.
10. Resuspend the washed resin in 2 bed-volumes of degassed labeling buffer containing 200 µM 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide (BTFMA) and incubate the mixture for 6 h at 4°C with gentle agitation.
11. Decant and resuspend the TALON resin in fresh labeling buffer containing 200 µM BTFMA and incubate the mixture for another 6 h to overnight at 4°C with gentle agitation. 
12. Load the A_2AR-bound TALON resin in a gravity column. Wash the resin with 10 column-volumes of LMNG storage buffer, followed by 4 column-volumes of LMNG storage buffer containing 20 mM imidazole.
13. Elute A_2AR with 5 column-volumes of LMNG storage buffer containing 250 mM imidazole.
14. Run SDS-PAGE gel at this point to check receptor yield and purity.
15. Remove imidazole by buffer exchanging the sample to LMNG storage buffer using a 50,000 Da MWCO spin-concentrator. Concentrate the sample to 5-10 mL, then add the sample to XAC-agarose gel (Affi-Gel 10 conjugated with the A_2AR antagonist xanthine amine congener (Weiß and Grisshammer, 2002; Ye et al., 2018)) washed with LMNG storage buffer at a 1:1 sample-to-gel ratio. Incubate overnight at 4 °C with gentle agitation.
16. Load the A_2AR-bound XAC resin in a gravity column. Wash the resin with 1 column-volume of LMNG storage buffer followed by 1 column-volume of cholate storage buffer. (Note: washing with more than 1 column-volume of cholate buffer will begin to elute A_2AR off the XAC resins)
17. Elute A_2AR with 3 column-volumes of XAC elution buffer containing 30 mM theophylline.
18. Run SDS-PAGE gel to check receptor yield and purity.
19. Prior to nanodisc assembly, buffer-exchange the sample to cholate storage buffer using a 30,000 Da MWCO spin-concentrator, such that the final theophylline concentration is <100 µM. Concentrate the receptor to a final concentration of ~50 µM as determined by a nanodrop spectrophotometer, using the extinction coefficient of A_2AR at 280 nm and blanking with the flow-through from the spin-concentrator. Note: This concentration will be an overestimation since the sample may not be pure at this stage, and that A_2AR-bound theophylline also absorbs at 280 nm. Receptor concentration can also be estimated from an SDS-PAGE gel by comparing the A_2AR band intensities against a sample of known concentration. 

Nanodisc assembly and purification

The final nanodisc reconstitution mixture contains 15 mM cholate, 100 µM MSPΔH5, and a 3.5 mM total lipid concentration which in turn is composed of a 3:2 ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The 35:1 lipid:MSPΔH5 ratio has been optimized in our lab for producing monodisperse receptor-embedded nanodiscs using the current protocol and may need to be re-optimized for different proteins or conditions. The reconstitution volume is determined by the volume of the receptor sample (which may contain excess cholate from rounds of spin-concentration) as well as the A_2AR concentration prior to nanodisc assembly. Typically, we aim for a reconstitution volume that is at least 5 times that of the receptor sample while keeping the receptor concentration below 10% that of the MSPΔH5. For example, if 1 mL of 50 µM A_2AR (in cholate storage buffer) is obtained after the XAC column, one will require a total reconstitution volume of at least 5 mL, where the final A_2AR concentration is 10 µM. The first requirement prevents the addition of excess cholate, while the second requirement reduces the chance of having more than one copy of A_2AR in each nanodisc. Table 1 provides a summary of the ingredients and their amounts needed for such a reconstitution mixture.

Table 1. Recipe for a 5 mL nanodisc reconstitution

1. Using glass or chloroform-resistant syringes, transfer chloroform-solubilized POPC and POPG into a glass scintillation vial. Note: Lipids can be purchased as powders or chloroform-solubilized stocks.
2. In a fume hood, evaporate the chloroform under a gentle stream of nitrogen or argon gas. When most of the chloroform is removed, evaporate any residual solvent in a vacuum desiccator for 30 min. Cover the vial to protect the lipids from oxidizing light.
3. Dissolve the dried lipids in HNE buffer containing 100 mM sodium cholate. Incubate the vial at room temperature with gentle agitation until the lipids are completely solubilized.
4. Dilute the cholate with an appropriate amount of HNE buffer (e.g., Table 1), then place the vial on ice for 5 min.
5. Add the receptor and the MSPΔH5. Incubate the mixture on ice for 1 h. 
6. Wash Bio-Beads SM-2 resin (0.6 g/mL) with Ultrapure water in a gravity column. Apply gentle pressure to the column to remove excess water trapped in the resin.
7. Incubate the reconstitution mixture with the rinsed Bio-Beads at 4 °C for 5 h with gentle agitation. Remove the Bio-Beads using a gravity column and concentrate the nanodisc sample to ~3 mL.
8. Purify the sample using a HiLoad 16/600 Superdex 200 prep grade size exclusion column equilibrated with sterile and degassed nanodisc storage buffer at 4 °C and a flow rate of 1 mL/min.
9. Check the eluted fractions with SDS-PAGE. Monomeric nanodiscs typically elute at around 65 mL. This peak contains both A_2AR-embedded and empty nanodiscs.
10. Pool the fractions containing A_2AR-embedded nanodiscs. The edges of the peak are usually excluded to obtain a narrow size distribution. 
11. Incubate the nanodisc sample with TALON resin for 2 h at 4 °C, then wash the resin extensively with nanodisc storage buffer to remove empty (non-His-tagged) nanodiscs. 
12. Elute A_2AR-embedded nanodiscs with nanodisc storage buffer containing 250 mM imidazole
13. Buffer-exchange the sample to the desired buffer for downstream experiments. Receptors can be flash-frozen in nanodisc storage buffer and stored at -80 °C.

Recipes

YPD plates: 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 1.5% agar, 0.5 mg/mL G418

YPD medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 0.2 mg/mL G418

BMGY medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogenase base (YNB) without amino acids, 100 mM sodium phosphate (pH 6.5), 0.4 mg/L biotin, 1% (v/v) glycerol, 50 µg/mL kanamycin, 0.1 mL/L antifoam A

BMMY medium (without methanol): 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB without amino acids, 0.4 mg/L biotin, 0.4 g/L histidine, 2% (v/v) DMSO, 5 mM theophylline, 100 µg/mL ampicillin, 50 µg/mL kanamycin or 100 µg/mL ampicillin, 0.1 mL/L antifoam A

MSP Lysis buffer: 40 mM Tris/HCl, pH 8.0, 100 mM NaCl, 20 mg lysozyme, 1 ug/mL DNase I, 2 mM MgCl₂, 5 mM 6-aminocaproic acid, 5 mM benzamidine, 1 mM PMSF, 1% Triton X-100

MSP wash buffer A: 40 mM Tris/HCl, pH 8.0, 300 mM NaCl, 1% Triton X-100

MSP wash buffer B: 40 mM Tris/HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole

MSP elution buffer: 40 mM Tris/HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole

TEV cleavage buffer: 40 mM Tris/HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 5% glycerol

Cholate storage buffer: 50 mM HEPES, pH 7.4, 100 mM NaCl, 15 mM sodium cholate

Cell washing buffer: 50 mM HEPES, pH 7.4, 10% glycerol, 4 mM theophylline

A_2AR lysis buffer: 50 mM HEPES, pH 7.4, 300 mM NaCl, 4 mM theophylline, 5 mM 6-aminocaproic acid, 5 mM benzamidine, 10% glycerol

Membrane solubilization buffer: 50 mM HEPES, pH 7.4, 300 mM NaCl, 4 mM theophylline, 1 mM 6-aminocaproic acid, 1 mM benzamidine, 0.75% LMNG, 0.075% CHS

Labeling buffer: 50 mM HEPES, pH 7.4, 300 mM NaCl, 0.05% LMNG, and 0.005% CHS

LMNG storage buffer: 50 mM HEPES, pH 7.4, 300 mM NaCl, 0.05% LMNG

XAC elution buffer: 50 mM HEPES, pH 7.4, 100 mM NaCl, 15 mM sodium cholate, and 30 mM theophylline

Nanodisc storage buffer: 50 mM HEPES, pH 7.4, 100 mM NaCl

HNE buffer: 20 mM HEPES, pH 8.0, 100 mM NaCl, 0.5 mM EDTA

HNE buffer with 100 mM cholate: 20 mM HEPES, pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 100 mM sodium cholate

References

Effendi WI, Nagano T, Kobayashi K, Nishimura Y. 2020. Focusing on Adenosine Receptors as a Potential Targeted Therapy in Human Diseases. Cells 9:785. doi:10.3390/cells9030785

Guerrero A. 2018. A2A Adenosine Receptor Agonists and their Potential Therapeutic Applications. An Update. Curr Med Chem 25:3597–3612.

Hagn F, Etzkorn M, Raschle T, Wagner G. 2013. Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135:1919–1925. doi:10.1021/ja310901f

Huang SK, Almurad O, Pejana RJ, Morrison ZA, Pandey A, Picard L-P, Nitz M, Sljoka A, Prosser RS. 2022. Allosteric modulation of the adenosine A2A receptor by cholesterol. Elife 11:e73901. doi:10.7554/eLife.73901

Huang SK, Pandey A, Tran DP, Villanueva NL, Kitao A, Sunahara RK, Sljoka A, Prosser RS. 2021. Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling. Cell 184:1884-1894.e14. doi:10.1016/j.cell.2021.02.041

Ruiz MDL, Lim Y, Zheng J. 2014. Adenosine A2A Receptor as a Drug Discovery Target. J Med Chem 57:3623–3650.

Weiß HM, Grisshammer R. 2002. Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli. Eur J Biochem 269:82–92. doi:10.1046/j.0014-2956.2002.02618.x

Ye L, Orazietti AP, Pandey A, Prosser RS. 2018. High-efficiency expression of yeast-derived G-protein coupled receptors and 19F labeling for dynamical studies In: Ghose R, editor. Protein NMR. Methods in Molecular Biology. New York: Humana Press. pp. 407–421. doi:10.1007/978-1-4939-7386-6

Ye L, Van Eps N, Zimmer M, Ernst OP, Scott Prosser R. 2016. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature 533:265–268. doi:10.1038/nature17668

Yu F, Zhu C, Xie Q, Wang Y. 2020. Adenosine A2A Receptor Antagonists for Cancer Immunotherapy. J Med Chem 170. doi:10.1021/acs.jmedchem.0c00237

Zheng J, Zhang X, Zhen X. 2019. Development of Adenosine A2A Receptor Antagonists for the Treatment of Parkinson’s Disease: A Recent Update and Challenge. ACS Chem Neurosci 10:783–791. doi:10.1021/acschemneuro.8b00313