Jul 31, 2023

Public workspaceEXPRESSION AND PURIFICATION OF HUMAN NEMO (GST-GFP-NEMO)

DOI
dx.doi.org/10.17504/protocols.io.261gedpojv47/v1
  • 1Max Perutz Labs, University of Vienna, Vienna Biocenter, Vienna, Austria;
  • 2Vienna Biocenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria
OLIVIA HARDING
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DOI: dx.doi.org/10.17504/protocols.io.261gedpojv47/v1
Protocol Citation: Elisabeth Holzer 2023. EXPRESSION AND PURIFICATION OF HUMAN NEMO (GST-GFP-NEMO). protocols.io https://dx.doi.org/10.17504/protocols.io.261gedpojv47/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 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83006
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)
Abstract
This protocol describes how to express and purify human NEMO (IKK-γ) tagged N-terminally with GST and EGFP. The expression is performed with E. coli Rosetta pLysS cells. The protein is purified via a GST batch purification and gel filtration (SEC).
Materials
pGEX-GST-Thrombin-TEV-GFP-NEMO (Addgene ID: 199781)
E. coli Rosetta (DE3) pLysS cells
LB medium with antibiotics: 50 μl/ml ampicillin and 34 μl/ml chloramphenicol
IPTG (Isopropyl-β-D-thiogalactopyranosid)
Thrombin (Serva, #36402.01)

Columns/Resin:
Glutathione Sepharose 4B (Cytiva, #17075605)
Superose 6 increase 10/300 column (Cytiva, #17517201)
Before start
Lysis Buffer:
50 mM HEPES pH 7.5
300 mM NaCl
2 mM MgCl2
Freshly added: 2 mM β-Mercaptoethanol, Roche Protease Inhibitor (Merck, #5056489001), and DNAse I (Sigma, #DN25-1G)

Wash Buffer 1:
50 mM HEPES pH 7.5
300 mM NaCl
Freshly added: 1 mM DTT

Wash Buffer 2:
50 mM HEPES pH 7.5
700 mM NaCl
Freshly added: 1 mM DTT

SEC Buffer:
25 mM HEPES pH 7.5
150 mM NaCl
Freshly added: 1 mM DTT


Expression
Expression
16h 15m
16h 15m
Grow E. coli Rosetta (DE3) pLysS cells in Amount2 L LB medium at Temperature37 °C until achieving an OD600 nm of 0.4.

Reduce temperature to Temperature18 °C and continue growth to OD600 nm of 0.8.

Induce protein expression with Concentration250 micromolar (µM) IPTG and continue growth for ~Duration16:00:00 at Temperature18 °C

16h
Centrifuge the cells at 3000 rcf (Temperature4 °C ,Duration00:15:00 )

15m
Aspirate media and resuspend cell pellet in ~ 10 ml of 1X PBS, centrifuge the cells at 3000 rcf (Temperature4 °C ,Duration00:15:00 ) and take off the supernatant.
15m
Flash freeze the pellet in liquid nitrogen and store at Temperature-80 °C until use.

Purification
Purification
46m 30s
46m 30s
Thaw pellet and resuspend in Lysis Buffer with freshly added Concentration2 millimolar (mM) β-Mercaptoethanol, Roche Protease Inhibitor and DNAse I

Sonicate sample Duration00:00:30 at 65% power for 5 cycles (Bandelin Sonopuls)

1m 30s
Repeat sonication for a total of 3X
Centrifuge at 48000 rcf at Temperature4 °C for Duration00:45:00

45m
During this step, equilibrate Amount3 mL Glutathione Sepharose 4B beads by washing the slurry with water and Wash Buffer 1 (~ 6 ml).
Filter supernatant after centrifugation through a Thikness0.45 µm syringe filter

Incubate the sample on equilibrated beads on a tube roller at Temperature4 °C for Duration04:00:00

4h
Wash beads 5X with Wash Buffer 1
Wash beads 1X with Wash Buffer 2

Note
These wash steps remove nonspecific proteins

Wash beads 2X with Wash Buffer 1
Note
These washes remove high salt concentration

Cleave the resulting sample to produce GFP-NEMO by incubating with thrombin DurationOvernight at Temperature4 °C

1d
Centrifuge beads at 3000 rcf for Duration00:03:00 at Temperature4 °C

3m
Collect supernatant and filter through Thikness.45 µm syringe filter

Concentrate product to a final volume of Amount500 µL using a 10 kDa MWCO concentrator

The concentrated and filtered protein can be applied onto a Superose 6 increase column (10/300 Cytiva) pre-equillibrated with SEC buffer
Note
This step can remove contamination and degradation products

Fractions containing the purified proteins are pooled, concentrated, frozen in liquid nitrogen and stored at - 80°C
Note
Protein purity can be determined by SDS PAGE analysis and measurement of protein concentration