TITLE: Purification of BCL2L13-GFP
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of BCL2L13-GFP.

[STEPS]
SECTION: Purification - BCL2L13-GFP
1. To purify GFP-tagged 

BCL2L13-GFP (available from Addgene), 
BCL2L13(W276A/I279A)-GFP (ΔLIR1) (available from Addgene), 
BCL2L13(Y213A/I216A/W276A/I279A)-GFP (ΔLIR1+2) (available from Addgene),
BCL2L13(I224A/L227A/W276A/I279A)-GFP (ΔLIR1+3)(available from Addgene),
BCL2L13(W276A/I279A/I307A/V310A)-GFP (ΔLIR1+4) (available from Addgene),
BCL2L13(I224A/L227A/W276A/I279A/I307A/V310A)-GFP (ΔLIR1+3+4)(available from Addgene), 

fuse the cytosol-exposed domain of BCL2L13 (1-463aa) to a C-terminal GFP-tag through cloning into a pET-DUET1 vector (available on Addgene).
SECTION: Purification - BCL2L13-GFP
2. Introduce the point mutants by in vitro mutagenesis to generate
 
BCL2L13 W276A/I279A (ΔLIR1)(available on Addgene), 
BCL2L13 Y213A/I216A/W276A/I279A (ΔLIR1+2) (available on Addgene), 
BCL2L13 I224A/L227A/W276A/I279A (ΔLIR1+3) (available on Addgene), 
BCL2L13 W276A/I279A/I307A/V310A (ΔLIR1+4) (available on Addgene), 
BCL2L13 I224A/L227A/W276A/I279A/I307A/V310A (ΔLIR1+3+4) (available on Addgene). 
SECTION: Purification - BCL2L13-GFP
3. After the transformation of the pET-DUET1 vector encoding BCL2L13-GFP wild-type or mutants in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow the cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continued at 18 °C. 
SECTION: Purification - BCL2L13-GFP
4. Once the cells reaches an OD600 of 0.8, induce the protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. Collect the cells by centrifugation and resuspend in lysis buffer. 
 
Lysis buffer:

 
 
SECTION: Purification - BCL2L13-GFP
5. Sonicate the cell lysates twice for 30 s and clear by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 
SECTION: Purification - BCL2L13-GFP
6. 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 BCL2L13-GFP. 
SECTION: Purification - BCL2L13-GFP
7. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification - BCL2L13-GFP
8. Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

 Wash buffer:

 
  
High salt wash buffer:

 
 
SECTION: Purification - BCL2L13-GFP
5.1. Sonicate the cell lysates for 30 s(1/2).
SECTION: Purification - BCL2L13-GFP
5.2. Sonicate the cell lysates for 30 s(2/2).
SECTION: Purification - BCL2L13-GFP
9. To cleave off the GST-tag  ,  elute the GFP-tagged cargo receptor from the GSH beads by the addition of TEV protease.
SECTION: Purification - BCL2L13-GFP
10. To collect the supernatant, collect the beads by centrifugation. 
SECTION: Purification - BCL2L13-GFP
11. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
SECTION: Purification - BCL2L13-GFP
12. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 50 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 
SECTION: Purification - BCL2L13-GFP
13. Elute the proteins with SEC buffer. 

 SEC buffer:  

 
 
SECTION: Purification - BCL2L13-GFP
14. Analyze fractions by SDS-PAGE and Coomassie staining. Pool fractions containing purified BCL2L13-GFP. 
SECTION: Purification - BCL2L13-GFP
15. After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.