TITLE: Purification of GST-WIPI1/WIPI2d/WIPI3/WIPI4
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of GST-tagged WIPI1/2d/3/4.

[STEPS]
SECTION: Purification procedure
1. To purify GST-WIPI1/GST-WIPI2/GST-WIPI3/GST-WIPI4, we express the GST-tagged WIPI1/2d/3/4 from a pCAG backbone encoding GST-TEV-WIPI1/2/3/4 (available from Addgene). 

SECTION: Purification procedure
23. Store the proteins at -80 °C.

SECTION: Purification procedure
14. 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
12. 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 GST-TEV-WIPI1/2/3/4. 
 
SECTION: Purification procedure
11. Clear the cell lysates by centrifugation at 10000 rpm, 45 min, 4 °C with a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

SECTION: Purification procedure
10. Sonicate the cell lysates twice for 30 s. 

SECTION: Purification procedure
9. For purification of GST-TEV-WIPI1/2/3/4, resuspend the cell pellet in, (complete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma), and DNase (Sigma)). 

25ml lysis buffer:
 
 
SECTION: Purification procedure
8. Store the pellets at -80 °C. 

SECTION: Purification procedure
7. Wash the pellet with PBS to remove medium and then flash-frozen in liquid nitrogen. 

SECTION: Purification procedure
6. Another 1440 min later, harvest the cells by centrifugation at 270 x g, 20 min. 

SECTION: Purification procedure
5. One day post transfection, supplement the culture with100 mLEXCELL R 293 Serum-Free Medium (Sigma-Aldrich, 14571C- 1000ML). 

SECTION: Purification procedure
4. On the day of transfection, transfect a 400 mL culture with 400 1671 of the MAXI-prep DNA, diluted in 13 mL of Opti-MEMR I Reduced Serum Medium (Thermo, 31985-062), and 800 1671Polyethylenimine (PEI 25K, Polysciences CatNo 23966-1), also diluted in 13 mL of Opti-MEM media. 

SECTION: Purification procedure
3. The day before transfection, seed the cells at a density of 0.7 x 10∧6 cells per ml. 

SECTION: Purification procedure
2. Express the protein in FreeStyleTM HEK293F cells, grown at 37 °C in FreeStyle™ 293 Expression Medium (Thermo, 12338-026). 

SECTION: Purification procedure
13. Centrifuge the samples to pellet the beads and remove the unbound lysate. 

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

SECTION: Purification procedure
15. Incubate the beads 120 min with 4 mL of  50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C, to elute GST-tagged WIPI1/2/3/4 from the beads. 

Wash buffer: 
 
 
SECTION: Purification procedure
21. Pool the fractions containing purified GST-TEV-WIPI1/2/3/4. 

SECTION: Purification procedure
20. Analyse the fractions by SDS-PAGE and Coomassie staining.
SECTION: Purification procedure
16. To collect the supernatant, collect the beads by centrifugation. 

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

SEC buffer:
 
 
SECTION: Purification procedure
18. Pool and filterate the supernatant fractions through a 0.45 µm syringe filter, concentrate with 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 

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