Aug 29, 2024

Public workspacePurification of BCL2L13-GFP

DOI
dx.doi.org/10.17504/protocols.io.36wgqno2ogk5/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
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DOI: dx.doi.org/10.17504/protocols.io.36wgqno2ogk5/v1
Protocol Citation: Elias Adriaenssens 2024. Purification of BCL2L13-GFP. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqno2ogk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 24, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 103090
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol details the purification of BCL2L13-GFP.
Materials
Lysis buffer:
AB
Tris-HCl50 mM
pH 7.4
NaCl300 mM
Triton X-1001%
glycerol5%
MgCl22 mM
DTT1 mM
β-mercaptoethanol2mM
cOmplete EDTA-free protease inhibitors (Roche)
CIP protease inhibitor (Sigma)
DNase (Sigma)
Wash buffer:

Tris-HCl 50 mM
pH7.4
NaCl300 mM
DTT1 mM
High salt wash buffer:

Tris-HCl 50 mM
pH7.4
NaCl700 mM
DTT1 mM
SEC buffer:
Tris-HCl 25 mM
pH7.4
NaCl300 mM
DTT1 mM
  • pET-DUET1 vector (available on Addgene)ReagentpETDuet-1 TIM9,10addgeneCatalog #170280
  • 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)
  • Rosetta pLysS cells (Novagen Cat# 70956-4)ReagentRosetta™(DE3)pLysS Competent Cells - NovagenMerckCatalog #70956-4
  • SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific)
  • Glutathione Sepharose 4B beads (GE Healthcare)
  • 10 kDa cut-off Amicon filter (Merck Millipore)ReagentAmicon® Ultra Centrifugal Filter, 10 kDa MWCOMerck MilliporeSigma (Sigma-Aldrich)Catalog #UFC801008
  • GFP-tagged BCL2L13-GFP (available from Addgene),
  • BCL2L13(W275A/I278A)-GFP (ΔLIR1) (available from Addgene),
  • BCL2L13(Y213A/I216A/W275A/I278A)-GFP (ΔLIR1+2) (available from Addgene),
  • BCL2L13(I224A/L227A/W275A/I278A)-GFP (ΔLIR1+3)(available from Addgene),
  • BCL2L13(W275A/I278A/I307A/V310A)-GFP (ΔLIR1+4) (available from Addgene),
  • BCL2L13(I224A/L227A/W275A/I278A/I307A/V310A)-GFP (ΔLIR1+3+4)(available from Addgene)


Purification - BCL2L13-GFP
Purification - BCL2L13-GFP
20h 46m
20h 46m
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).
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).
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 Temperature37 °C until an OD600 of 0.4 and then continued at Temperature18 °C .
Temperature
Once the cells reaches an OD600 of 0.8, induce the protein expression with Concentration100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for Duration16:00:00 at Temperature18 °C . Collect the cells by centrifugation and resuspend in lysis buffer.
Lysis buffer:

AB
Tris-HCl pH 7.450 mM
NaCl300 mM
Triton X-1001%
Glycerol5%
MgCl22 mM
DTT1 mM
β-mercaptoethanol2mM
cOmplete EDTA-free protease inhibitors (Roche)
CIP protease inhibitor (Sigma)
DNase (Sigma)
16h
Centrifigation
Temperature
Sonicate the cell lysates twice for 30 s and clear by centrifugation at Centrifigation18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Centrifigation
Temperature
Sonicate the cell lysates for Duration00:00:30 (1/2).
30s
Sonicate the cell lysates for Duration00:00:30 (2/2).
30s
Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for Duration02:00:00 at Temperature4 °C with gentle shaking to bind BCL2L13-GFP.
2h
Incubation
Temperature
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Centrifigation
Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.

Wash buffer:

AB
Tris-HCl pH 7.450 mM
NaCl300 mM
DTT1 mM
High salt wash buffer:

AB
Tris-HCl pH 7.450 mM
NaCl700 mM
DTT1 mM
Wash
To cleave off the GST-tag DurationOvernight , elute the GFP-tagged cargo receptor from the GSH beads by the addition of TEV protease.
Incubation
Overnight
Temperature
To collect the supernatant, collect the beads by centrifugation.
Centrifigation
Wash the beads twice with Amount4 mL of wash buffer, and collect the supernatant.
Wash
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).
Elute the proteins with SEC buffer.

SEC buffer:

AB
Tris-HCl pH 7.425 mM
NaCl300 mM
DTT1 mM
Analyze fractions by SDS-PAGE and Coomassie staining. Pool fractions containing purified BCL2L13-GFP.
After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.

Note
Store the proteins at Temperature-80 °C .

Temperature