byJoanna Geddes, Chris Wojewodzki, Barbara Kaboord, and Kay Opperman
G protein-coupled receptors (GPCRs), spanning 7-transmembrane domains, are a large protein family of receptors that play a key role in transmitting signals across the cell membrane. The requirement for stabilized G protein-coupled receptors (GPCRs) in their native, functional form presents significant challenges for in vitro functional and structural characterization. Since endogenous GPCR expression levels are typically very low, most researchers elect to overexpress their GPCR of interest in order to obtain the sample concentration needed for structural analysis. There are many considerations when choosing your expression system. While E. coli and insect expression systems may yield more protein, it has been shown that mammalian systems are more ideal to mimic native conformation of receptors for functional and structural analysis [1,2]. Based on the expression system chosen, adjustments to the extraction protocol are required to get the most efficient solubilization of functional receptor. Maintaining solubility and native conformation of overexpressed GPCR constructs throughout the downstream purification process is also challenging. Therefore, optimization of the affinity purification protocol and incorporation of stabilization factors into the buffer systems are required to maintain receptor functionality throughout the sample preparation [3,4,5].
Here we report an optimized method of extraction and purification of adenosine receptor type 2A (A2AR) that yields purified receptor ready for functional analysis. Extraction of A2AR using the GPCR Extraction and Stabilization Reagent yields a stable receptor in whole cell lysate that can be stored for up to 1 week at 4⁰C or 1 month at -20⁰C. The extracted receptor can then be processed further using affinity purification appropriate for the tagged target. The addition of the GPCR Extraction and Stabilization Reagent diluted 10-fold into the recommended wash and elution buffers aids in maintaining receptor stability throughout the entire purification process.
Whole Cell Protein Extraction: Fresh or frozen pelleted Expi293 cells (1x107) expressing C-terminally His-tagged A2AR were washed in 1 mL PBS and pelleted at 500 x g for 5 minutes. After the supernatant was discarded, the cell pellet was subsequently suspended in 1 mL of cold GPCR Extraction and Stabilization Reagent (Cat No. A43436) by either gently pipetting up and down, using a dounce homogenizer (25–50 strokes), or sonicating at 30% Amps for 10 seconds to lyse. Cells were then incubated for 60 minutes at 4ºC with end-over-end mixing. After incubation, samples were centrifuged at 16,000 x g for 20 minutes at 4ºC to pellet cell debris. The supernatant containing the solubilized protein was collected. Protein concentration was determined using the Pierce Rapid Gold BCA Protein Assay Kit (Cat No. A53225).
Pierce Ni-NTA Magnetic Agarose Beads (Cat No. 78605) (100 µL of a 25% slurry) were pipetted into a 1.5 mL microcentrifuge tube for each sample. The beads were then washed twice with 500 µL of cold Equilibration Buffer (25 mM HEPES, 0.3 M NaCl, 10 mM imidazole, mixed 9:1 with GPCR Extraction and Stabilization Reagent, pH 8.0). Protein extract (1 mL diluted to 1 mg/mL with Equilibration Buffer) was added to the washed beads, vortexed for 10 seconds, and then mixed on an end-over-end rotator for 60 minutes at 4ºC. After incubation, the beads were collected on a magnetic stand, and the supernatant was saved (flow-through) for downstream analysis. Next, the beads with the bound A2AR-GFP-His were washed twice with 500 µL of Wash Buffer (25 mM HEPES, 0.3 M NaCl, 15 mM imidazole, mixed 9:1 with GPCR Extraction and Stabilization Reagent, pH 8.0). Bound receptor was eluted from the beads by adding 250 µL of Elution Buffer (25 mM HEPES, 0.3 M NaCl, 0.3 M imidazole, mixed 9:1 with GPCR Extraction and Stabilization Reagent, pH 8.0) to the washed beads and then incubated on an end-over-end rotator for 10 minutes at room temperature.
Note: Thermostability may vary by receptor, depending on your target, an elution at 4ºC with increased incubation time may be necessary. Beads were then collected on a magnetic stand, and the eluate containing A2AR-GFP-His was carefully removed. Samples were either directly analyzed by SDS-PAGE and Silver Stain or buffer exchanged into the GPCR Extraction and Stabilization Reagent for overnight storage at 4ºC using Thermo Scientific Zeba Spin Desalting Columns before proceeding to radio-ligand binding assays.
A2AR-GFP-His starting lysate, flow-through, and purified samples were prepared from frozen Expi293 cell pellets. For the binding assay, 5 μg of each sample were incubated for 75 minutes at room temperature with either 3H-Adenosine alone to determine total binding, or with unlabeled adenosine and 3H-Adenosine to determine non-specific binding. Free radioligand was then removed using Pierce Dye and Biotin Removal Spin Columns, and bound radioligand was then quantified using a TRI-CARB 2000 TR scintillation counter.
Figure 1: When comparing different homogenization methods for extraction of functional A2AR, the following trends were observed:
1) Increased agitation improved protein yield.
2) Dounce homogenized samples yielded the highest amount of active A2AR.
3) Sonication increased protein yield but decreased the amount of active receptor.
Figure 2: The ability to purify A2AR-GFP-His in the presence of GPCR Extraction and Stabilization Reagent at 1/10 strength successfully stabilized the receptor throughout the process.
Figure 3: Our purification protocol demonstrated enriched activity of A2AR-GFP-His in the final eluate. When comparing equal amounts of starting protein load (5 µg) to the eluate samples, there is a 4-fold increase in activity following purification.
Using agitation at the beginning of the lysis incubation will increase protein yield; however, too much, as in the case with sonication, affects the conformation of the target protein. We recommend using gentle agitation at this step using a dounce or handheld tissue homogenizer depending on sample volume (Figure 1). The addition of GPCR Extraction and Stabilization Reagent at 1/10 strength into the purification buffer system does not negatively influence the binding of A2AR-GFP-His to the Ni-NTA magnetic agarose beads (Figure 2). This addition is important to keep the critical micelle concentration constant as it plays a crucial role in maintaining functionality of A2AR-GFP-His throughout the entire purification process. This is demonstrated when comparing specific activity of equal amounts of starting lysate, flow-through, and eluates. A significant enrichment (>4-fold) of purified active receptor is observed (Figure 3). With evolving techniques and technologies, such as native mass spectrometry and cryo-EM, extraction of a stable receptor will be essential. With the improved receptor stability that the GPCR Extraction and Stabilization Reagent provides in both the extraction and purification methods, researchers will have a greater flexibility and confidence in their GPCR research and targeted therapeutic studies.
For Research Use Only. Not for use in diagnostic procedures.