TITLE: Purification of BCL2L13-GST
AUTHORS: Elias Adriaenssens

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

[STEPS]
SECTION: Purification - BCL2L13-GST
1. To purify BCL2L13-GST, fuse the cytosol-exposed domain of BCL2L13 (1-463aa) to a C-terminal GST-tag through cloning into a pET-DUET1 vector (available on Addgene). 
SECTION: Purification - BCL2L13-GST
2. Introduce the point mutants by in vitro mutagenesis to generate 
BCL2L13 W275A/I278A (ΔLIR1)(available on Addgene), 
BCL2L13 Y213A/I216A/W275A/I278A (ΔLIR1+2) (available on Addgene), 
BCL2L13 I224A/L227A/W275A/I278A (ΔLIR1+3) (available on Addgene), 
BCL2L13 W275A/I278A/I307A/V310A (ΔLIR1+4) (available on Addgene), 
BCL2L13 I224A/L227A/W275A/I278A/I307A/V310A (ΔLIR1+3+4) (available on Addgene). 
SECTION: Purification - BCL2L13-GST
3. After the transformation of the pET-DUET1 vector encoding BCL2L13-GST 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-GST
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-GST
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-GST
5.1. Sonicate the cell lysates for 30 s(1/2).
SECTION: Purification - BCL2L13-GST
5.2. Sonicate the cell lysates for 30 s(2/2).
SECTION: Purification - BCL2L13-GST
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-GST. 
SECTION: Purification - BCL2L13-GST
7. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification - BCL2L13-GST
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-GST
9. Incubate the beads 120 min with 4 mL of 50 millimolar (mM) reduced glutathione dissolves in wash buffer at 4 °C, to elute BCL2L13-GST from the beads. 
SECTION: Purification - BCL2L13-GST
10. To collect the supernatant, collect the beads by centrifugation. 
SECTION: Purification - BCL2L13-GST
11. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 

SECTION: Purification - BCL2L13-GST
12. 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). 
SECTION: Purification - BCL2L13-GST
13. Elute the proteins with SEC buffer. 

 SEC buffer:  

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