TITLE: Purification of mCherry-ATG101/13 (1-191aa) subcomplex
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of mCherry-ATG101/13 (1-191aa) subcomplex.

[STEPS]
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
1. To purify mCherry-ATG13/101 HORMA dimer, we express mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (available from Addgene) together with GST-TEV-ATG101 (available from Addgene). 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
2. Express the ATG13/101 HORMA dimer in FreeStyleTM HEK293F cells, grow at 37 °C in FreeStyle™ 293 Expression Medium (Thermo, 12338-026). 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
3. The day before transfection, seed the cells at a density of 0.7 x 10^6 cells per ml. 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
4. On the day of transfection, transfect a 400 mL culture with 400 µg of plasmid at a molar 1:1 ratio, dilute in 13 mL of Opti-MEMR I Reduced Serum Medium (Thermo, 31985-062), and 800 µgPolyethylenimine (PEI 25K, Polysciences CatNo 23966-1), also dilute in 13 ml of Opti-MEM media. 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
5. One day post transfection, supplement the culture with 100 mL EXCELL R 293 Serum-Free Medium (Sigma-Aldrich, 14571C- 1000ML). 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
6. Another 24 h later, harvest the cells by centrifugation at 270 x g, 20 min. 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
7. Wash the pellet with PBS to remove medium and then flash-frozen in liquid nitrogen. 

 

SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
8. For purification of the ATG13/101 subcomplex, resuspend the cell pellet in 25 mL lysis buffer. 

Lysis buffer:
 
 
 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
9. Homogenize the cells with a douncer and clear the lysates by centrifugation at 10000 x g, 45 min, 4 °C with a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
10. 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 GST-TEV-ATG101/mCherry-ATG13(1-191aa). 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
11. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
12. Wash the beads twice with wash buffer I followed by three washes in wash buffer II. 

Wash buffer I: 

 
 
Wash buffer II:
 
 
 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
13. Incubate the beads 120 min with TEV protease in wash buffer at 4 °C, to release mCherry- or GFP-tagged ATG13/101 from the beads. 

Wash buffer:
  
 
 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
14. To collect the supernatant, collect the beads by centrifugation.
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
15. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
16. 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 Superose S6 Increase 10/300 GL column (Cytiva). 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
17. Elute the proteins with SEC buffer. 

SEC buffer:
 
 
 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
18. Analyze the fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing both ATG13/101. 
SECTION: Purification - mCherry-ATG101/13 (1-191aa) subcomplex
19. After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.