TITLE: Purification GFP-ATG13 IDR
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of GFP-ATG13 IDR.

[STEPS]
SECTION: Purification
1. To purify GFP-tagged ATG13 IDR, the coding sequence for ATG13 (190-517aa), (206-517aa), (231-517aa), (190-205_231-517aa), (190-230aa), (190-205aa), or (206-230aa) into GST-TEV-EGFP-insert through cloning into a pGEX-4T1 vector (Plasmids available from Addgene). 
SECTION: Purification
2. Mutants 

3A (M196A/S197A/R199A) and 
11A (M196A/S197A/R199A/G202A/T204A/P205A/I207A/M208A/I210A/D213A/H214A) 

are also expressed according to the protocol below.
SECTION: Purification
3. After the transformation of the pGEX-4T1 vectors encoding the GFP-tagged ATG13 IDR in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C. 
SECTION: Purification
4. Once the cells reaches an OD600 of 0.8, induce the protein expression with 100 micromolar (µM) isopropyl β-D-1-t isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. 
SECTION: Purification
5. Collect cells by centrifugation and resuspend in lysis buffer for GST-tagged proteins (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 2 mM MgCl2, 5% glycerol, 1% Triton X-100, 2 mM β-mercaptoethanol, cOmplete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma), and DNase (Sigma)). 

Lysis buffer:

 
 
SECTION: Purification
6. Sonicate the cell lysates twice for 30 s. 
SECTION: Purification
7. Clear the lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 
SECTION: Purification
8. Collect the supernatant after centrifugation and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C on a roller to bind GST-TEV-EGFP-ATG13 IDR. 
SECTION: Purification
9. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification
10. 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
11. Incubate the beads 120 min with TEV protease at 4 °C, to elute GFP-tagged ATG13 IDR from the beads. 
SECTION: Purification
12. To collect the supernatant, collect the beads by centrifugation. 
SECTION: Purification
13. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
SECTION: Purification
14. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrated with 10 or 30 kDa cut-off Amicon filter (Merck Millipore). 
SECTION: Purification
15. Load the samples onto a pre-equilibrated Superose 200 Increase 10/300 GL column (Cytiva) or S75 Increase 10/300 column (Cytiva) in case of the smaller peptides (190-230aa and smaller variants thereof). 
SECTION: Purification
16. Elute the proteins with SEC buffer. 

SEC Buffer:

 
 
SECTION: Purification
17. Analyze the fractions by SDS-PAGE and Coomassie staining. 
SECTION: Purification
18. Pool the fractions containing purified ATG13 IDR. After concentrating the purified protein, aliquote the protein was and snap-frozen in liquid nitrogen. 
SECTION: Purification
19. Store the proteins at -80 °C.