Mar 19, 2025
Purification of Recombinant Human Hsc70 from Escherichia coli
- Patricia Yuste-Checa1,
- Nadine Wischnewski1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany
Protocol Citation: Patricia Yuste-Checa, Nadine Wischnewski, F Ulrich Hartl 2025. Purification of Recombinant Human Hsc70 from Escherichia coli. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqd2pkvk5/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: March 11, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124242
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to efficiently purify recombinant human heat shock cognate 71 kDa protein (Hsc70), also known as HSPA8, expressed as hexahistidine-tagged, TEV protease cleavable fusion protein in Escherichia coli.
Materials
Buffers:
- Lysis buffer:
| A | B | |
| HEPES-KOH pH 8.0 | 50 mM | |
| KCl | 10 mM | |
| MgCl2 | 5 mM |
- Elution buffer:
| A | B | |
| HEPES-KOH pH 8.0 | 50 mM | |
| KCl | 10 mM | |
| MgCl2 | 5 mM | |
| Imidazole | 1000 mM |
- High salt buffer:
| A | B | |
| HEPES-KOH pH 8.0 | 50 mM | |
| KCl | 1000 mM | |
| MgCl2 | 5 mM |
- Size exclusion chromatography (SEC) buffer:
| A | B | |
| HEPES-KOH pH 7.5 | 50 mM | |
| KCl | 150 mM | |
| MgCl2 | 5 mM | |
| Glycerol | 5% |
Hsc70 Expression: Transformation
Hsc70 Expression: Transformation
9h 37m 45s
9h 37m 45s
Thaw RbCl-competent Escherichia coli Bl21 cells (DE3) On ice .
Add 1 µL of pProEx-HtA Hsc70 plasmid and incubate 00:30:00 On ice .
30m
Heat shock 00:00:45 at 42 °C .
45s
Incubate On ice 00:02:00 , then add 850 µL Lysogeny broth (LB) or Super Optimal broth with Catabolite repression (SOC) medium.
2m
Shake for 01:00:00 at 37 °C .
1h
Centrifuge for 3000 x g, 00:05:00 and remove most of the supernatant.
5m
Resuspend the pellet with the remaining supernatant and plate the bacteria on LB containing 100 µL Ampicillin agar plates and incubate Overnight at 37 °C .
8h
Hsc70 Expression: Bacterial Culture
Hsc70 Expression: Bacterial Culture
18h 45m
18h 45m
Prepare preculture: Scrape all colonies with the scraper and inoculate 25 mL -50 mL LB containing 100 µL Ampicillin in a flask. Shake flask at 37 °C for 04:00:00 -06:00:00 .
6h
Measure OD600 of the preculture and inoculate 6 L of LB medium containing 100 µL Ampicillin (6 flasks) to an OD600 = 0.05.
Shake flasks at 37 °C until an approximate OD600 of 0.5-0.8 (02:00:00 -04:00:00 ).
4h
Add isopropyl β-D-1-thiogalactopyranoside (IPTG) at final concentration of 0.5 millimolar (mM) .
Shake flasks Overnight at 21 °C .
8h
Centrifuge bacterial culture at 4000 rpm, 00:45:00 in a JS-4.2 rotor (Beckman). Discard supernatant.
45m
Resuspend each pellet with Lysis buffer (20 mL per 1 L bacterial culture) supplemented with Complete EDTA-free protease inhibitor cocktail (Merck). Flash-freeze in liquid nitrogen for storage at -70 °C .
Hsc70 Purification: Lysis
Hsc70 Purification: Lysis
1h 15m
1h 15m
Thaw the cell pellets in a water bath at 22 °C and add Lysis buffer (75 mL per 2 L bacteria) supplemented with Complete EDTA-free protease inhibitor cocktail (Merck), Sm DNase 50 U mL-1 and 1 µL lysozyme and incubate under gentle shaking for 00:30:00 at 4 °C .
30m
Lyse the cells with a Emulsiflex C5 high-pressure homogenizer (4 cycles at 20,000 psi).
Centrifuge lysate at 40000 rpm, 4°C, 00:45:00 . Keep clarified supernatant.
45m
Ni-NTA Chromatography
Ni-NTA Chromatography
Equilibrate the Ni-NTA column (HisTrap HP column 5 mL; 17-5248-0, Cytiva) with 10 column volumes (CV, 15 mL) Lysis buffer.
Load lysate supernatant onto the Ni-NTA column (50 mL).
Wash the Ni-NTA column with 2 CV 99% Lysis buffer - 1% Elution buffer.
Wash the Ni-NTA column with 2 CV 95% Lysis buffer - 5% Elution buffer.
Elute His6-TEV-Hsc70 with 10 CV gradient 5-50% Elution buffer and collect elution fractions.
Wash the Ni-NTA column with 2 CV gradient 50-100% Elution buffer.
Wash the Ni-NTA column with 1 CV gradient Elution buffer.
Analyze fractions by SDS-PAGE and Coomassie blue staining.
Ni-NTA chromatogram and SDS-PAGE analysis. P: 20 µL resuspended pellet + 20 µL 2x SDS PAGE sample buffer, loaded 15 µL. L: 20 µL lysate + 20 µL 2x SDS-PAGE sample buffer, loaded 15 µL. Fractions of interest: 10 µL + 10 µL 2x SDS-PAGE sample buffer; loaded 6 µL. Red boxes: Pooled elution fractions (Fractions 29-40, 30 mL).
Desalting
Desalting
In order to exchange the buffer, load the eluted protein onto a HiPrep 26/10 desalting column (17-5087-01, Cytiva) equilibrated with 2 CV (1 CV, 55 mL) of Lysis buffer.
Load 15 mL of the collected elution fractions. Run several times if needed.
Wash the column with 2 CV of High salt buffer.
His-TEV Protease Cleavage
His-TEV Protease Cleavage
8h
8h
Collect fractions eluted from desalting column containing protein and add His-TEV protease at final concentration of 400 U per mg of protein.
Incubate at 4 °C Overnight .
Note
If precipitation is observed, reduce the salt concentration to 150 millimolar (mM) KCl by dilution.
8h
Ni-NTA Filter Column (Collect Flow Through)
Ni-NTA Filter Column (Collect Flow Through)
Equilibrate the Ni-NTA column with Lysis buffer.
Load the cleavage mixture onto the column.
Collect the flow through where the cleaved Hsc70 protein should elute.
Wash the column with 3 CV 95% Lysis buffer - 5% Elution buffer.
Wash the column with 3 CV 75% Lysis buffer - 25% Elution buffer.
Wash the column with 3 CV Elution buffer and collect eluted fractions.
Analyze fractions of the flow-through by SDS-PAGE and Coomassie blue staining.
Ni-NTA chromatogram and SDS-PAGE analysis. -/+ TEV: 10 µL sample + 10 µL 2x SDS PAGE sample buffer, loaded 2.0 µL. Fractions of interest: 10 µL sample + 10 µL 2x SDS PAGE sample buffer, loaded 10 µL. Red box: Pooled flow-through fractions (Fractions 3-12, 55 mL).
Anion Exchange Chromatography
Anion Exchange Chromatography
Equilibrate a MonoQ column (MonoQ HR10/10, 17-0506-01 Cytiva, CV: 8 mL) with 2 CV of Lysis buffer.
Load the pooled protein from the Ni-NTA column flow-through.
Wash with 2 CV of Lysis buffer.
Elute the protein with a gradient of 15 CV 1-70% High salt buffer.
Wash the column with a gradient of 2 CV 70-100% High salt buffer.
Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.
MonoQ chromatogram and SDS-PAGE analysis. Fractions of interest: 10 µL sample + 10 µL 2x SDS-PAGE sample buffer, loaded 10 µL. Red box: Pooled fractions (Fractions 33-44, 28 mL).
Concentrate the pooled fractions using a centrifugal ultrafiltration device (Sartorius VivaSpin 10 kDa) to 5 mL volume for size exclusion chromatography.
Size Exclusion Chromatography
Size Exclusion Chromatography
Load the concentrated sample onto a Sephacryl S100 16/60 column (17-1165-01, Cytiva) previously equilibrated with SEC buffer.
Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
Size exclusion chromatogram and SDS-PAGE analysis. 10 µL fraction of interest + 10 µL 2x SDS-PAGE sample buffer, loaded 15 µL. Collected eluted fractions (24-36, 13 mL).
Pool fractions containing Hsc70, concentrate to around 10 µL , aliquot and flash-freeze in liquid nitrogen for storage at -70 °C .