TITLE: Purification of CK2 kinase complex
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of CK2 kinase complex.

[STEPS]
SECTION: Purification procedure
1. To purify the CK2 kinase complex, subclone GST-TEV-CK2α together with CK2β in a pFastBac-Dual vector (available from Addgene) and GST-TEV-CK2α’ together with CK2β in a pFastBac-Dual vector (available from Addgene) for co-expression in insect cells. 

SECTION: Purification procedure
2. Use the constructs to generate bacmid DNA, using the Bac-to-Bac system, by amplification in DH10BacY cells 25. 

SECTION: Purification procedure
3. After the bacmid DNA was verified by PCR for insertion of the transgene, purify bacmid DNA for transfection into Sf9 insect cells (12659017, Thermo Fisher, RRID:CVCL_0549). 

SECTION: Purification procedure
4. To this end, mix 2500 ng of plasmid DNA with FuGene transfection reagent (Promega) and transfected 1 million Sf9 cells seeded in a 6 well plate. 

SECTION: Purification procedure
5. About 7 days after transfection, harvest the V0 virus and use to infect 40 mL of 1 million cells per ml of Sf9 cells. 

SECTION: Purification procedure
6. Closely monitor the viability of the cultures and upon the decrease in viability and confirmation of yellow fluorescence, collect the supernatant after centrifugation and stored this as V1 virus. 

SECTION: Purification procedure
7. For expressions, infect 1 L of Sf9 cells (12659017, Thermo Fisher, RRID:CVCL_0549), at 1 million cells per ml, with 1 mL of V1 virus for GST-TEV-CK2α/CK2β and 1 mL of V1 virus for GST-TEV-CK2α’/CK2β. 

SECTION: Purification procedure
8. When the viability of the co-infected cells decreased to 90-95%, collect cells by centrifugation. 

SECTION: Purification procedure
9. Wash the cell pellets with 1x PBS and flash-frozen in liquid nitrogen. 

SECTION: Purification procedure
10. Store pellets at -80 °C. 

SECTION: Purification procedure
11. For purification of the CK2 kinase complex, resuspend pellet  in 25 ml lysis buffer 1 µl benzonase (Sigma), complete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma)). 
 
 25ml Lysis buffer:
 
 
SECTION: Purification procedure
12. Homogenize and clear the cells with a douncer and lysates  by centrifugation at 18000 rpm, 45 min, 4 °C  in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

SECTION: Purification procedure
13. Collect and incubate the supernatant with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind the CK2 complex. 

SECTION: Purification procedure
14. Centrifuge samples to pellet the beads and remove the unbound lysate.
SECTION: Purification procedure
15. Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

Wash buffer:
 
 
Salt wash buffer:
 
 
SECTION: Purification procedure
16. Incubate beads 120 min with TEV protease in wash buffer at 4 °C. 

Wash buffer:
 
 
SECTION: Purification procedure
17. After  release the proteins from the beads by the TEV protease, collect the supernatant after centrifugation of the beads. 

SECTION: Purification procedure
18. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 

SECTION: Purification procedure
19. Pool and filter the supernatant fractions through a 0.45 µm syringe filter, and concentrated with a 10 kDa cut-off Amicon filter (Merck Millipore). 

SECTION: Purification procedure
20. Load the proteins onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 

SECTION: Purification procedure
21. Elute the proteins with SEC buffer. 

SEC buffer:
 
 
SECTION: Purification procedure
22. Analyze fractions by SDS-PAGE and Coomassie staining. 

SECTION: Purification procedure
23. Pool the fractions containing purified CK2α/CK2α’/CK2β. 

SECTION: Purification procedure
24. After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen.