TITLE: Purification of BNIP3-GST
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of BNIP3-GST.

[STEPS]
SECTION: Purification - BNIP3-GST
1. To purify BNIP3-GST, we purchase the gene-synthesized codon-optimized cytosol-exposed domain of BNIP3 (1-158aa) fused to a C-terminal GST-tag in a pFastBac-Dual vector from Genscript (available from Addgene). 
SECTION: Purification - BNIP3-GST
2. Introduce the point mutants by in vitro mutagenesis to generate BNIP3 E44A/L47A/D49A/A50K/Q51A (5A; ΔWIPI2) (available from Addgene), and BNIP3 W18A/L21A (ΔLIR) (available from Addgene). 
SECTION: Purification - BNIP3-GST
3. The constructs are used to generate bacmid DNA, using the Bac-to-Bac system, by amplification in DH10BacY cells. 
SECTION: Purification - BNIP3-GST
4. After verifying the bacmid DNA by PCR for insertion of the transgene, we purifiy bacmid DNA for transfection into Sf9 insect cells (12659017, Thermo Fisher, RRID:CVCL_0549). 
SECTION: Purification - BNIP3-GST
5. To this end, we mix 2500 ng of plasmid DNA with FuGene transfection reagent (Promega) and transfect 1 million Sf9 cells seeded in a 6 well plate. 
SECTION: Purification - BNIP3-GST
6. About 7 days after transfection, harvest the V0 virus and used to infect 40 ml of 1 million cells per ml of Sf9 cells. 
SECTION: Purification - BNIP3-GST
7. Closely monitor the viability of the cultures and upon the decrease in viability and confirmation of yellow fluorescence, we collects the supernatant after centrifugation and store this as V1 virus. 
SECTION: Purification - BNIP3-GST
8. For expressions, we infect 1 L of Sf9 cells, at 1 million cells per ml, with 1 ml of V1 virus. 
SECTION: Purification - BNIP3-GST
9. When the viability of the cells decreases to 90-95%, collect the cells by centrifugation. 
SECTION: Purification - BNIP3-GST
10. Wash the cell pellets with 1x PBS and flash-frozen in liquid nitrogen. 

 

SECTION: Purification - BNIP3-GST
11. For purification of BNIP3-GST wild-type or mutants, resuspend the pellets in 25 ml lysis buffer.
 
Lysis buffer:

 
 
SECTION: Purification - BNIP3-GST
12. Cells were homogenized with a douncer and clear the cell lysates by centrifugation at 18.000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 
SECTION: Purification - BNIP3-GST
13. Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind BNIP3-GST. 
SECTION: Purification - BNIP3-GST
14. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification - BNIP3-GST
15. Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

Wash buffer:
 
 
 
High salt buffer:
 
 
 
SECTION: Purification - BNIP3-GST
16. Incubate the beads 120 min with 4 mL of 50 millimolar (mM) reduced glutathione dissolves in wash buffer at 4 °C, to elute BNIP3-GST from the beads. 
SECTION: Purification - BNIP3-GST
17. To collect the supernatant, collect the beads by centrifugation. 
SECTION: Purification - BNIP3-GST
18. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
SECTION: Purification - BNIP3-GST
19. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). Elute the proteins with SEC buffer. 

 SEC buffer:

 
 
SECTION: Purification - BNIP3-GST
20. Analyze the fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified BNIP3-GST. 
SECTION: Purification - BNIP3-GST
21. After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.