TITLE: Purification of BNIP3-GFP
AUTHORS: Elias Adriaenssens

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

[STEPS]
SECTION: Purification - BNIP3-GFP
1. To purify GFP-tagged 

BNIP3-GFP (available from Addgene) or BNIP3(W18A/L21A)-GFP (ΔLIR) (available from Addgene), 

we purchase the gene-synthesized codon-optimized cytosol-exposed domain of BNIP3 (1-158aa) fused to a C-terminal GFP-tag in a pFastBac-Dual vector from Genscript (available from Addgene). 
SECTION: Purification - BNIP3-GFP
2. Introduce the point mutants by in vitro mutagenesis to generate 

BNIP3 W18A/L21A (ΔLIR) (available from Addgene). 
SECTION: Purification - BNIP3-GFP
3. The constructs are used to generate bacmid DNA, using the Bac-to-Bac system, by amplification in DH10BacY cells. 
SECTION: Purification - BNIP3-GFP
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-GFP
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-GFP
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-GFP
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-GFP
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-GFP
9. When the viability of the cells decreases to 90-95%, collect the cells by centrifugation. 
SECTION: Purification - BNIP3-GFP
10. Wash the cell pellets with 1x PBS and flash-frozen in liquid nitrogen. 

 

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

 
 
SECTION: Purification - BNIP3-GFP
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-GFP
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-GFP. 
SECTION: Purification - BNIP3-GFP
14. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification - BNIP3-GFP
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-GFP
16. Cleave off the GST-tag   by eluting the GFP-tagged cargo receptor from the GSH beads by the addition of TEV protease in wash buffer at 4 °C.
SECTION: Purification - BNIP3-GFP
17. To collect the supernatant, collect the beads by centrifugation. 
SECTION: Purification - BNIP3-GFP
18. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
SECTION: Purification - BNIP3-GFP
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-GFP
20. Analyze the fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified BNIP3-GFP. 
SECTION: Purification - BNIP3-GFP
21. After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.