Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells

The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is then purified in a buffer containing dodecyl maltoside using Strep-Tactin affinity chromatography. Following enzymatic cleavage of the attached GFP and Strep-tag by thrombin, the P2X7 receptor is isolated using size exclusion chromatography. This method typically yields ~2 mg of purified protein from 6 L of Sf9 culture. Purified protein can be stored with a buffer containing 15% glycerol at 4 °C for at least 2 months and used for a variety of functional and structural studies (Karasawa and Kawate, 2016).


SEC buffer (see Recipes)
Equipment

A.
Sf9 culture (perform in a tissue culture hood except for centrifugation steps)

1.
Sf9 cells in a cryo-vial are stored in liquid nitrogen.

2.
Thaw an aliquot (1-2 ml) of stock Sf9 insect cells (stored in liquid nitrogen) in a 37 °C water bath and resuspend into 10 ml Sf-900 III medium.

3.
Spin down the cells at 125 × g for 5 min, remove the medium by pipetting, and gently resuspend the cells in a 15 ml conical tube with 10 ml Sf900 III medium by pipetting up and down.

4.
Transfer the cells into a 250 ml flask and add 15 ml of Sf900 III to make a 25 ml cell suspension.

5.
Incubate the cells in a temperature controlled shaker at 27 °C and at 125 rpm.

6.
Monitor the cell density every day by manually counting the number of cells using a hemocytometer.

8.
Incubate the cells in a temperature controlled shaker at 27 °C and at 125 rpm.

9.
After repeating the steps A6-A8 for 2-3 times, the doubling time should become approximately 24 h. Maintain Sf9 cultures at 0.5-3.5 × 10 6 cell/ml in a 250 ml flask and grow up in a larger flask for P2 virus infection.

B.
Preparation of bacmids

1.
The pNGFP-FB3 vector ( Figure 1) harbors STREP tag, EGFP, thrombin recognition site, multiple cloning site (MCS) and a stop codon. The P2X7 receptor gene is subcloned between BamHI and XhoI sites.

3.
Heat shock the cells for 45 sec at 42 °C and put them on ice for 2 min.

4.
Add 500 μl of SOC medium into the cells and shake in a temperature controlled incubator at 225 rpm at 37 °C for 4 h.

5.
Harvest the cells by centrifugation at 20,000 × g for 1 min and plate them on a LB-Bac plate (see Recipes) (add ~50 μl SOC medium if necessary).

6.
Incubate the LB-Bac plate for 2 days at 37 °C.

7.
Pick up a white colony (Figure 2), inoculate 8 ml of LB-Bac medium, and culture in a temperature controlled incubator at 225 rpm at 37 °C overnight.

8.
Harvest the cells by centrifugation at 2,500 × g for 10 min.

10.
Add 200 μl of 'Solution 2' in Plasmid mini kit (Omega Bio-Tek) and mix by flipping the tube several times.

11.
Add 270 μl of 'Solution 3' in Plasmid mini kit (Omega Bio-Tek) and mix by flipping the tube several times.

12.
Harvest the bacmid containing solution by centrifugation at 20,000 × g for 10 min (save supernatant) and place it into a new 1.5 ml tube.

13.
Add 700 μl of phenol-chloroform and mix well by vortexing the tube for a few seconds.

14.
Centrifuge at 20,000 × g for 2 min and transfer the upper layer ( Figure   3) into a new 1.5 ml tube.

15.
Add 700 μl of chloroform and mix well by vortexing for a few seconds.

16.
Centrifuge at 20,000 × g for 2 min and transfer the upper layer into a 2.0 ml tube.

17.
Add 1.4 ml of 100% ethanol and mix well by vortexing for a few seconds.

19.
Harvest the bacmid by centrifugation at 20,000 × g for 15 min at 4 °C.

21.
Aspirate the 70% ethanol and dry the bacmid containing pellet in a tissue culture hood for 30 min.

C.
Virus production

4.
Incubate the mixture for 15 min at room temperature and add to the wells of the previously plated 6-well plate by pipetting dropwise.

6.
Collect the P1 virus containing media and filter sterilize using a 0.22 μm filter. Store the P1 virus at 4 °C.

8.
Culture the cells in a temperature controlled incubator at 125 rpm for 3 days at 27 °C.

9.
Collect the P2 virus containing media by centrifugation at 2,500 × g for

5.
Break the cells by nitrogen cavitation using a cell disruption vessel (600 psi for 20 min; see Videos 1 and 2). Vessel should be incubated at 4 °C after nitrogen loading.

7.
Transfer the supernatant into three 70 ml ultracentrifuge tubes and balance them using the resuspension buffer. It is critical to balance the centrifuge tubes precisely and to fill it up to the shoulder of the tubes (Figure 7).

11.
Set the homogenizer at ~900 rpm and homogenize the membrane fraction with five strokes going up and down (see Video 4).

12.
Solubilize the homogenized membrane fraction with ~350 ml of the solubilization buffer (see Recipes) by stirring at 300 rpm for 1 h.

13.
Transfer the supernatant into six 70 ml ultracentrifuge tubes and balance them using the solubilization buffer.

15.
Pool the supernatant (Figure 9) into a 500 ml beaker. Add 6 ml of Strep-Tactin resin pre-equilibrated with the solubilization buffer, and stir at 200 rpm for 60 min.

16.
Harvest the resin by centrifugation at 2,500 × g for 5 min in a 50 ml tube and transfer into two 25 ml gravity columns.

17.
Wash the resin with 20 ml of the washing buffer (see Recipes) for each column.

19.
Concentrate the eluted GFP-P2X7 down to 1 ml using a 100 kDa-cutoff spin column. Measure the protein concentration using a spectrophotometer.

21.
Remove aggregated protein by centrifugation at 264,360 × g for 10min.

22.
Inject the protein into the Superdex 200 column and collect the peak fractions.

Data analysis
Quality of the purified P2X7 receptor can be analyzed by SEC and SDS-PAGE ( Figure 11). Figure 11A shows a representative SEC profile with a single P2X7 peak, suggesting that the purified P2X7 receptor is monodisperse. A representative SDS-PAGE gel image ( Figure  11B) verifies the chemical purity of this sample.

1.
Even a slightly higher concentration of gentamicin may be too toxic to DH10Bac cells. Use it exactly at 6.7 μg/ml.

3.
P2 virus should be always prepared fresh. Noticeable reduction of P2X7 receptor expression is observed with the usage of more than one week old virus.

4.
Temperature shift from 27 °C to 18 °C increases the expression level of P2X7 by more than four fold.

7.
Inclusion of glycerol in both elution and SEC buffers is necessary for avoiding aggregation of P2X7.

Polyethylenimine
Dissolve 1 g polyethylenimine in 900 ml MilliQ water Adjust the pH to 7.0 with NaOH Volume up to 1 L with MilliQ water