Jan 13, 2025

Public workspacePurification of CHMP2B

DOI
dx.doi.org/10.17504/protocols.io.bp2l623r1gqe/v1
  • Cole Sitron1,
  • Victoria A Trinkaus1,
  • Nadine Wischnewski1,
  • Carolin Klose2,
  • F Ulrich Hartl2
  • 1Max Planck Institute of Biochemistry;
  • 2Technical University of Munich
Cole Sitron
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DOI: dx.doi.org/10.17504/protocols.io.bp2l623r1gqe/v1
Protocol Citation: Cole Sitron, Victoria A Trinkaus, Nadine Wischnewski, Carolin Klose, F Ulrich Hartl 2025. Purification of CHMP2B. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l623r1gqe/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: June 10, 2024
Last Modified: January 13, 2025
Protocol Integer ID: 103396
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000282
Aligning Science Across Parkinson’s
Grant ID: ASAP- 024268
Abstract
This protocol describes a method for purification of recombinant CHMP2B from E Coli. To improve solubility, CHMP2B is tagged with His-SUMO and expressed at a low temperature. The His-tagged SUMO fusion is first isolated on a nickel column before cleavage of the SUMO tag and a reverse nickel column to separate His-SUMO from the cleaved CHMP2B. The CHMP2B is then cleaned up by size exclusion.
Materials
• BL21 (DE3) E Coli
• LB media (VWR cat. no. J106-2 kg)ReagentLB BrothAmrescoCatalog #J106-2KG
• Terrific Broth media (Sigma cat. no. T0918)ReagentTerrific Broth, modifiedMerck MilliporeSigma (Sigma-Aldrich)Catalog #T0918
• Kanamycin sulfate (Sigma cat. no. K4000-50G)ReagentKanamycin sulfate from Streptomyces kanamyceticusMerck MilliporeSigma (Sigma-Aldrich)Catalog #K4000-50G
• IPTG (1M stock in water) (Roth cat. no. CN08.3)ReagentIPTG, 50 gCarl RothCatalog #CN08.3
• pET28-His-SUMO-CHMP2B plasmid
• Incubator capable of Temperature18 °C and Temperature37 °C
• Tip sonicator
• Nanodrop or other spectrophotometer capable of measuring 220 nm absorbance
• FPLC
• PBS (Thermo Fisher Scientific ca. no. 20012068)ReagentPBS, pH 7.2Thermo FisherCatalog #20012068
• Lysis Buffer:
AB
Tris50 mM
pH8
NaCl500 mM
Imidazole10 mM
β-Mercaptoethanol (β-ME)2 mM
glycerol5 % (v/v)
cOmplete Protease Inhibitor Cocktail (Roche cat. no. 11873580001)1x

ReagentcOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001

Note
β-ME stock is 14.3 M.

• Nickel Buffer 1A:
Tris50 mM
pH8
NaCl500 mM
β-ME2 mM
• Nickel Buffer 1B:
AB
Tris50 mM
pH8
NaCl500 mM
Imidazole500 mM
β-ME2 mM
• Dialysis Buffer:
AB
Tris20 mM
pH7.5
NaCl150 mM
Imidazole4 mM
β-ME2 mM
• Nickel Buffer 2A: 20 mM Tris pH 7.0, 150 mM NaCl, 2 mM β-ME
AB
Tris20 mM
pH7
NaCl150 mM
β-ME2 mM
• Nickel Buffer 2B: 20 mM Tris pH 7.0, 150 mM NaCl, 500 mM Imidazole, 2 mM β-ME
AB
Tris20 mM
pH7
NaCl150 mM
Imidazole500 mM
β-ME2 mM
• SEC Buffer: 20 mM Tris pH 7.0, 150 mM NaCl, 2 mM β-ME
AB
Tris20 mM
pH7
NaCl150 mM
β-ME2 mM
• Glycerol
• His-SenP2 protease (50 U/μl, Max Planck Institute of Biochemistry Core Facility)
• Superdex S200 16/60 pg column (GE cat. no. 28989335)ReagentGE Hiload 16/60 Superdex 200 PgGE HealthcareCatalog #28989335
• 3 Ni-NTA HisTrap Columns (Cytiva cat. no. 17524802)Reagent3 Ni-NTA HisTrap HP, 5 x 5 mlCytiva Life SciencesCatalog #17524802
• Vivaspin® 20, 10 kDa MWCO concentration spin column (Sigma cat. no. GE28-9323-60)ReagentVivaspin® 20, 10 kDa MWCO PolyethersulfoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #GE28-9323-60
• PES Syringe Filter 0.22 μm (Merck Millipore cat. no. SLGP033RB)ReagentMillex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone, 33 mm, gamma sterilizedMerckCatalog #SLGPR33RB
• PES Vacuum Filter 0.22 μm (Corning cat. no. 431097)ReagentCorning® 500 mL Vacuum Filter/Storage Bottle System, 0.22 µm Pore 33.2cm² PES Membrane, Sterile, 12/CorningCatalog #431097
• 3.5 kDa Dialysis Tubing (Spectrum cat. no. 132724)ReagentSpectra/Por 3 Dialysis Tubing 3.5 kD 45mm 50ftRepligen (Spectrum Labs)Catalog #132724

Culture Growth
Culture Growth
16h
16h
Transform BL21 (DE3) cells with pET28-His-SUMO-CHMP2B plasmid and grow an DurationOvernight culture in Amount50 mL LB media with Concentration0.05 mg/mL Kanamycin at Temperature37 °C .
Overnight
Temperature
On the next day, dilute the culture 1:100 and grow cells in Terrific Broth supplemented with Concentration0.05 mg/mL Kanamycin at Temperature30 °C until it reaches an OD600 of 0.3.
Temperature
Reduce the incubation temperature to Temperature18 °C .
Temperature
After the culture grows to an OD600 of 0.6, induce protein expression Concentration0.5 millimolar (mM) IPTG for Duration16:00:00 .
16h
Harvest cells and wash the pellet once with PBS.
Pipetting
Freeze the pellets in liquid nitrogen and store at Temperature-80 °C .
Temperature
Lysis
Lysis
1h 22m
1h 22m
Thaw cells TemperatureOn ice and resuspend (by pipetting) in lysis buffer. Use Amount50 mL lysis buffer per Amount1 L of culture.
Pipetting
Temperature
Lyse cells via sonication with a tip sonicator (Duration00:20:00 , Duration00:00:20 on, Duration00:01:40 off).
22m
Centrifuge lysate at Centrifigation40000 x g, 01:00:00 .

Note
Carefully check to ensure that all of the solid material sedimented and supernatant is clear.

1h
Centrifigation
Nickel Column
Nickel Column
Equilibrate 3 x HisTrap columns with 98% Nickel Buffer 1A with Concentration10 millimolar (mM) Imidazole (2% Nickel Buffer 1B).

Filter supernatant with the vacuum filter and apply to the column.
Wash with 5 column volumes of Nickel Buffer 1A with Concentration10 millimolar (mM) Imidazole (2% Nickel Buffer 1B).
Wash
Wash with 4 column volumes of Nickel Buffer 1A with Concentration50 millimolar (mM) Imidazole (10% Nickel Buffer 1B).
Wash
Elute with 5 column volumes of Nickel Buffer 1B (Concentration500 millimolar (mM) Imidazole).
Check fractions by gel and pool the fractions with His-SUMO-CHMP2B.

Figure 1. SDS-PAGE analysis of fractions (numbers below) before loading and after running the first nickel column. His-SUMO-CHMP2B runs around 50 kDa. In this purification, fractions 45-48 were pooled. P, pellet.

SUMO Tag Cleavage and Reverse Nickel Column
SUMO Tag Cleavage and Reverse Nickel Column
1m 40s
1m 40s
Add Amount150 µL His-SenP2 protease and glycerol to 10% final to the eluate, then dialyze into Dialysis Buffer DurationOvernight .
1m 40s
Overnight
Filter the sample through a Amount0.22 µL syringe filter.
Perform reverse Ni-NTA affinity purification with Nickel Buffer 2A and 2B, this time collecting the flow-through instead of the eluate.

Note
The now untagged CHMP2B will be in the flow-through.

Check the fractions by gel and pool the CHMP2B-containing fractions.

Figure 2. SDS-PAGE analysis of fractions (numbers below) before loading and after running the second nickel column. -SenP represents the sample before cleavage sith SenP2, the cleaved fraction represents what was loaded onto the column, and the precipitate comes from a 20000 rpm spin in a Ti 45 rotor, shown to demonstrate the propensity to aggregate after cleavage. CHMP2B runs around 35 kDa. In this purification, fractions 8-14 were pooled.

Size Exclusion
Size Exclusion
Concentrate the CHMP2B to a final volume of Amount5 mL with a concentration spin column.
Load the protein onto a Superdex S200 16/60 pg column. Collect Amount5 mL fractions for the first Amount30 mL of SEC buffer, then collect Amount1 mL fractions for the next Amount75 mL of SEC buffer.
a. CHMP2B will begin to elute around Amount70 mL .



Figure 3. (Upper) Elution profile of cleaved CHMP2B on SEC. (Lower) SDS-PAGE gel of fractions before and after SEC. In this purification, fractions 54-60 were pooled.

Pipetting
Add 10% glycerol final to pooled fractions and concentrate the protein with a 10 kDa cutoff spin column to slightly more than Amount1.11 µL .

Note
a. CHMP2B has an extinction coefficient of 0. Concentration can be assessed Nanodrop at 220 undetermined or by a Bradford assay.
b. purified CHMP2B is now ready for aliquoting and snap freezing.

Pipetting