Expression and purification of recombinant SARS-CoV-2 Spike protein

Expression and Purification of Recombinant Full-length SARS-CoV-2 Spike Protein

Materials and Reagents

1. VWR ® 2L PC, Erlenmeyer Flask (VWR, catalog number: 10126-408)
2. Expi293F cell line (ThermoFisher, Gibco™, catalog number: A14527)
3. Expi293™ Expression Medium (ThermoFisher, Gibco™, catalog number: A1435101)
4. Penicillin-Streptomycin (ThermoFisher, Gibco™, catalog number: 15070063)
5. Puromycin (ThermoFisher, Gibco™ Puromycin Dihydrochloride, catalog number: A1113803)
6. FreeStyle™ 293 Expression Medium (FisherScientific, Gibco™, catalog number: 12-338-026)
7. 500 ml Corning® Centrifuge Tube (VWR, catalog number: 89091-000) 
8. Polyethylenimine (PEI, Polysciences, catalog number: 23966-1)
9. Tris (Millipore Sigma, catalog number: GE17-1321)
10. Sodium Chloride (Sigma-Aldrich, catalog number: S9888)
11. UltraPure™ 0.5M EDTA (ThermoFisher, Invitrogen™, catalog number: 15575020) 
12. Hydrochloric acid (HCL) (Sigma-Aldrich, catalog number: 320331)
13. Dulbecco's phosphate-buffered saline (DPBS) (Gibco, Catalog number:  14190144)
14. n-dodecyl-β-D-maltopyranoside (DDM) (Anatrace, catalog number: D310) 
15. NONIDET P- 40™ (NP-40) (AmericanBio, catalog number: AB01425-00500)
16. IBA LifeSciences Strep-Tactin Sepharose 50% suspension (FisherScientific, catalog number: NC0797617)
17. IBA LifeSciences Desthiobiotin (FisherScientific, catalog number: NC0395527)
18. Amicon® Ultra-15 Centrifugal Filter Units (Millipore Sigma, Catalog number: UFC910024)
19. Amicon Ultra-0.5 mL Centrifugal Filters (Millipore Sigma, Catalog number: UFC510096)
20. cOmplete™ Protease Inhibitor Cocktail (Millipore Sigma, SKU: 11836145001)
21. Dilution Buffer (see Recipes)
22. Cell Lysis Buffer (see Recipes)
23. Washing Buffers (see Recipes)
24. Elution Buffer (see Recipes)
25. Gel Filtration Buffer (see Recipes)

Equipment

1. ÄKTA purifier (GE Healthcare)
2. Fisherbrand™ Benchtop pH meter (Fisher Scientific) 
3. CO₂ Resistant Shaker (INFORS HT)
4. Biological safety cabinet (SterilGARD, Class II, Type A/B3)
5. Superose 6 Increase 10/300 GL (Cytiva)
6. Pump (Gilson mini plus 3)
7. Centrifuge (Beckman Coulter J6-MI)
8. Centrifuge (Beckman Coulter Avanti J-E)
9. Centrifuge (Beckman Coulter Alllegra X-14R)
10. PowerPac™ Basic Power Supply (Bio-Rad)
11. VWR nutating mixer (VWR)

Procedure

A. Prepare Expi293F Cells for Transfection

1. Grow 700 ml of 293F cells with Expi293 Expression Media supplemented with PenStrep and Puromycin (1ug/ml) in a 2L shaker flask.
2. Monitor the cell culture until the cell density reaches ~2.6 x 10⁶ with viability of ~95-98%. 

B. Transfection of Expi293F cells with Spike plasmid DNA (for 700 ml cell culture)

1. Dilute 700 mg of the full-length Spike plasmid DNA in 35 ml of FreeStyle 293F Medium.
2. Dilute 2.8 mg of PEI in 35 ml of FreeStyle 293F Medium. Incubate the two solutions at room temperature for 15 min.
3. Add the diluted PEI solution to the plasmid DNA solution, mix them well and incubate at room temperature for 15 min. The total volume should be ~70 ml.
4. Transfer 70 ml of the mixed solution into 700 ml Expi293F cells and incubate the cells in a shaker at 37°C with 80% relative humidity and 6.5% CO₂.

Note: The procedure can be scaled up or down proportionally for larger or smaller transfections. All the steps above should be performed in a biosafety level-2 cabinet.

C. Harvest the cells and purify the Spike protein

1. Transfer the cell culture to a 500 ml centrifuge tube and centrifuge at 1,000 xg for 30 min at 4°C, and then discard the supernatant.
2. Add 100 ml PBS to wash the cells, and centrifuge again at 1,000 xg for 15 min at 4°C, and discard the supernatant. 
3. Add 100-200 ml Lysis Buffer (see the recipes) supplemented with one tablet of protease inhibitor to the cell pellet.
4. Resuspend the cells completely and incubate with gently shaking for 1 hour at 4°C.
5. Transfer the cell lysate to a centrifuge tube, centrifuge at 30,000 xg for 1 hour at 4°C and collect the supernatant.
6. Equilibrate a Strep-Tactin column with Washing Buffer A (see the recipes), dilute the supernatant three folds with Dilution Buffer (see the recipes) and load it on the column using a peristaltic pump with a flow rate of  ~1 ml/min.
7. Wash the column sequentially with 50 column volumes each of Washing Buffer A, B and C (see the recipes). 
8. Elute the Spike protein with Elution Buffer (see the recipes) and analyze the fractions by SDS-PAGE.
9. Pool all fractions containing the Spike protein and concentrate to ~0.5 ml.
10. Load the concentrated sample on a Suprose 6 10/300 column with Gel Filtration Buffer (see the recipes) to further purify the protein.
11. Collect the peak fractions, concentrate and store the purified protein at -80°C.

Recipes

1. PEI Transfection Solution 
   Dissolve PEI powder to hot water at a final concentration of 1mg/ml, adjust pH of the solution to 7.0 at room temperature, and filter the solution with 0.22 um filter. 
2. Dilution Buffer
   100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA.
3. Cell Lysis Buffer
   100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and 1% (w/v) DDM.
4. Washing Buffer A
   100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and 0.3% (w/v) DDM.
5. Washing Buffer B
   100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and 0.1% (w/v) DDM.
6. Washing Buffer C
   100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and 0.02% (w/v) DDM.
7. Elution Buffer for strep-tactin column
   100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.02% (w/v) DDM and 5mM desthiobiotin.
8. Gel Filtration Buffer for Superose 6 10/300 column
   25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.02% (w/v) DDM

Note

Similar purification was also performed using detergent NP-40 instead of DDM. 

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