Feb 02, 2024
Purification of recombinant Low Density Lipoprotein Receptor Related Protein Associated Protein 1 (LRPAP1, RAP) from Escherichia coli
- Patricia Yuste-Checa1,
- Silvia Gärtner1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Protocol Citation: Patricia Yuste-Checa, Silvia Gärtner, F Ulrich Hartl 2024. Purification of recombinant Low Density Lipoprotein Receptor Related Protein Associated Protein 1 (LRPAP1, RAP) from Escherichia coli. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxpb2gx1/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: January 25, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94598
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to efficiently purify LDL Receptor Related Protein Associated Protein 1 (LRPAP1 or RAP) from Escherichia coli.
Attachments
Materials
Buffers
- Lysis buffer:
| A | B | |
| Tris-Cl pH 8.0 | 50 mM | |
| NaCl | 300 mM | |
| Imidazole | 10 mM |
- High salt buffer:
| A | B | |
| Tris-Cl pH 8.0 | 50 mM | |
| NaCl | 300 mM | |
| Imidazole | 250 mM |
- Low salt buffer:
| A | B | |
| Tris-Cl pH 8.0 | 50 mM | |
| NaCl | 10 mM |
- Size exclusion chromatography (SEC) buffer: 1x Phosphate buffered saline (PBS) pH 7.4
pQTEV-LRPAP1addgeneCatalog #31327
LRPAP1 express4ion
LRPAP1 express4ion
18h 37m 45s
Thaw RbCl-competent Escherichia coli Bl21 cells (DE3) On ice .
Add 1 µL of pQTEV-LRPAP1 plasmid without the signal peptide (1-35 amino acids) 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 /Ampicillin agar plates and incubate Overnight at 37 °C .
8h
Prepare preculture: Scrap all colonies with the scraper and inoculate 25-50 mL LB/Ampicillin. Shake at 37 °C for 4-6 h.
Measure OD600 of the preculture and inoculate 6 L of LB media to an OD600 = 0.05.
Shake flasks at 37 °C until approx. OD600 = 0.5-0.8. (2-4 h).
Add isopropyl β-D-1-thiogalactopyranoside (IPTG) at final concentration of 1 millimolar (mM) .
Shake flasks Overnight at 22 °C .
8h
Centrifuge bacterial culture at 4000 rpm, 01:00:00 . Discard supernatant.
1h
Resuspend each pellet with Lysis buffer (20 mL/1L bacteria) supplemented with Complete EDTA-free protease inhibitor cocktail (Merck). Flash-freeze in liquid nitrogen for storage at -80 °C .
Lysis
Lysis
1h 15m 50s
Thaw the cell pellets in a water bath at 22 °C and add lysis buffer (final volume 200 mL lysis buffer/ 6L bacteria) supplemented with Complete EDTA-free protease inhibitor cocktail (Merck) and Sm DNase 50 undetermined .
Add 1 undetermined lysozyme and incubate gently shaking for 00:30:00 at 4 °C .
30m
Sonicate lysate On ice , 8 cycles 00:00:20 ON, 00:00:30 OFF.
50s
Centrifuge lysate at 40000 rpm, 4°C, 00:45:00 .
45m
Ni-NTA chromatography
Ni-NTA chromatography
Equilibrate the Ni-NTA column with 10 column volumes (CV, 20 mL) Lysis buffer.
Load lysate supernatant to the Ni-NTA column.
Wash the Ni-NTA column with 10 CV Lysis buffer.
Elute His7-TEV-RAP with 5 CV 100% High salt buffer and collect elution fractions.
Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.
Ni-NTA chromatogram and SDS PAGE analysis. P: 20 µL resuspended pellet + 20 µL 2x SDS sample buffer, loaded 15 µL. S: 20 µL diluted supernatant + 20 µL 2x SDS sample buffer, loaded 15 µL. Fractions of interest: 10 µL + 10 µL 2x SDS sample buffer; loaded 6 µL. Green box: Collected elution fractions (Fractions 29-41, 65mL).
Desalting
Desalting
In order to reduce the salt concentration, load the eluted protein onto a HiPrep 26/10 desalting column equilibrated with the Low salt buffer.
His-TEV cleavage
His-TEV cleavage
8h
Collect eluted fraction containing protein and add glycerol at final concentration of 10%, DTT at final concentration of 1 millimolar (mM) , EDTA at final concentration of 0.25 millimolar (mM) and His-TEV at final concentration of 93U per mg of protein.
Incubate at 4 °C Overnight .
8h
Ni-NTA chromatography (Collect flow through)
Ni-NTA chromatography (Collect flow through)
Load the cleavage mixture onto a Ni-NTA column previously equilibrated with Lysis buffer and collect the flow through where the cleaved RAP protein should elute.
Wash the column with 5 CV (CV, 20 mL) of Lysis buffer and collect eluted fractions.
Analyze flow though fractions by SDS-PAGE and Coomassie blue staining.
Ni-NTA chromatogram and SDS PAGE analysis. TEV digest: 10 µL sample + 10 µL 2x SDS sample buffer, loaded 2.0 µL. Fractions of interest: 10 µL sample + 10 µL 2x SDS sample buffer, loaded 5 µL. Green boxes: Collected flow through (Fractions 20-21, 16 mL), and uncleaved His7-TEV-RAP protein eluted from the Ni-NTA column (Fractions 33-34).
Note
Some uncleaved His7-TEV-RAP protein may be eluted from the Ni-NTA column. Those fractions can be pooled, desalted and TEV digested again.
Size exclusion chromatography
Size exclusion chromatography
Load RAP-containing fractions onto a Superdex-200 column 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 sample dye, loaded 2.0 µL. Green box: Collected eluted fractions (72-77, 12mL).
Pool fractions containing RAP aliquot and flash-freeze in liquid nitrogen for storage at -80 °C .
Note
The protein can be concentrated with a filter device like a VivaSpin20, MWCO 10,000 Da. Approximate yield: from 6 L of bacterial culture around 95 mg of pure RAP are obtained.