Sep 04, 2024
Expression and purification of bacterial proteins (via N-terminal His-tag)
- Verena Dederer1,2,3,
- Sebastian Mathea1,2,3,
- Stefan Knapp1,2,3
- 1Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, Frankfurt 60438, Germany;
- 2Structural Genomics Consortium, Buchman Institute for Molecular Life Science (BMLS), Max-von-Laue-Straße 15, Frankfurt 60438, Germany;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: Verena Dederer, Sebastian Mathea, Stefan Knapp 2024. Expression and purification of bacterial proteins (via N-terminal His-tag). protocols.io https://dx.doi.org/10.17504/protocols.io.261ge514yg47/v1
Manuscript citation:
J Biol Chem. 2024 Jul;300(7):107469. doi: 10.1016/j.jbc.2024.107469. Epub 2024 Jun 12.
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: August 29, 2024
Last Modified: September 04, 2024
Protocol Integer ID: 106654
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
Abstract
Recombinant protein production in bacteria provides an efficient system for obtaining high yields in a short time and at low cost. Although not every protein is suitable for expression in bacteria, it is usually the first choice to start expression trials. If successful, more elaborate protein purification protocols can be developed. Here we provide a two-step protocol for protein purification from bacterial cells.
Materials
Transformation and protein expression
Chemically competent E.coli Rosetta cells
Plasmids encoding protein of interest
LB medium (lysogeny broth) - comercially available
LB agar plates (LB medium + 1% agar)
TB medium (terrific broth) - comercially available
Antibiotics: ampicilin, kanamycin, chloramphenicol
0.5 M Isopropyl-β-d-thiogalactopyranosid (IPTG)
Protein purification
Ni-NTA sepharose - comercially available
Lysis buffer (50 mM Hepes pH7.4; 500 mM NaCl; 20 mM imidazole; 0.5 mM TCEP; 5 % glycerol)
Elution buffer (50 mM Hepes pH7.4; 500 mM NaCl; 300 mM imidazole; 0.5 mM TCEP; 5 % glycerol)
S200 Superdex gel filtration column
Size-exclusion buffer (20 mM Hepes pH 7.4; 150 mM NaCl; 0.5 mM TCEP; 5% glycerol)
Liquid nitrogen
Safety warnings
This protocol requires handling of genetically modified organisms which requires an S1 facility.
Plamid Transformation into E.coli Rosetta cells
Plamid Transformation into E.coli Rosetta cells
1d 2h
1d 2h
Thaw 10 µL E.coli Rosetta cells gently on ice
20m
Add 25 ng respective expression plasmid and keep On ice for 00:30:00 min
♦ DARPin E11 in pQE30 vector (Qiagen) - this encodes: N-terminal 8xHis-tag and a C-terminal 3xFLAG-tag
♦ Rab8 in pET28A vector (Novagen) - this encodes a N‑terminal TEV‑cleavable 6xHis-tag
30m
Apply heatshock for 00:00:45 sec in a 42 °C waterbath
45s
Let cells recover for 00:05:00 min On ice
5m
Add 100 µL LB medium and incubate cells for 00:30:00 min at 37 °C
30m
Plate cell suspension on LB agar plates containing appropriate selection pressure
Incubate Overnight at37 °C
5m
Plates can be stored at 4 °C for several weeks or until further use
Large Scale Protein Expression (4L)
Large Scale Protein Expression (4L)
1d
1d
From a single colony of the LB plates inoculate50 mL LB medium containing the appropriate selection pressure
10m
Incubate the liquid culture shaking at 180 rpm, 37°C overnight
20h
Next morning, dilute grown overnight culture 10 mL into 1000 mL terrific broth expression medium containing the appropriate antibiotic
1h
Grow cells shaking at 180 rpm, 37°C until OD600 reached 1.0
Note
This may take about 04:00:00 h but regularly check OD600.
4h
Once OD600 reaches 1.0, reduce temperature to 18 °C and wait for another 00:30:00 min before adding 500 millimolar (mM) IPTG
30m
Incubate for 18:00:00 hours shaking at 180 rpm, 18°C
18h
Harvest cells by centrigufation with 1000 x g, 4°C, 00:15:00 min
25m
Freeze pellet and store -20 °C until further use
2-Step Protein Purification
2-Step Protein Purification
8h
8h
Thaw pellet and resuspend in 300 mL lysis buffer
Note
Lysis buffer contains
50 millimolar (mM) HEPES7.4 ,
500 millimolar (mM) NaCl
20 millimolar (mM) imidazole
0.5 millimolar (mM) TCEP
5 % (v/v) glycerol
20m
Lyse by sonication 3 rounds with 5 sec pulse, 10 sec pause, 3 min total pulse time On ice
30m
Clear lysate by ultracentrifugation 1000000 x g, 4°C, 00:45:00
1h
Perform Ni-NTA
1h
Load supernatant onto pre-equilibrated Ni-NTA sepharose beads
20m
Wash beads rigorously with lysis buffer (50 CV)
20m
Elute protein using lysis buffer supplemented with 300 millimolar (mM) imidazole
20m
Concentrate eluate to volume of 5 mL and subject to size-exclusion chromatography on a S200 gel filtration column
Note
size-exclusion buffer contains
20 millimolar (mM) HEPES 7.4
150 millimolar (mM) NaCl
0.5 millimolar (mM) TCEP
5 % (v/v) glycerol
3h
Pool fractions containing your protein of interest
30m
Measure protein concentration and do additional quality control measurements such as SDS PAGE with Coomassie staining, mass spectrometry to validate you sample
30m
Flash freeze aliquots of your protein of interest and store at -80 °C