Mar 27, 2025
Purification of Recombinant Amyloid Beta Peptide (M1-42) from Escherichia coli
- Patricia Yuste-Checa1,
- Nadine Wischnewski1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Protocol Citation: Patricia Yuste-Checa, Nadine Wischnewski, F Ulrich Hartl 2025. Purification of Recombinant Amyloid Beta Peptide (M1-42) from Escherichia coli. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4z2k2vo5/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 20, 2025
Last Modified: March 27, 2025
Protocol Integer ID: 125065
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol is based on Linse S. Methods Mol Biol. (2020), section “3.6 Purification of Aβ(M1–40) or Aβ(M1–42) without urea” and details how to efficiently purify Amyloid beta peptide (amino acids 1-42) with a starting methionine (Aβ, M1-42) from Escherichia coli inclusion bodies.
Materials
Buffers
- TE 7.5:
| A | B | |
| Tris-HCl pH 7.5 | 10 mM | |
| EDTA | 1 mM |
- TE 9.5:
| A | B | |
| Tris-HCl pH 9.5 | 10 mM | |
| EDTA | 1 mM |
- TE 8.5:
| A | B | |
| Tris-HCl pH 8.5 | 10 mM | |
| EDTA | 1 mM |
- TENa500:
| A | B | |
| Tris-HCl pH 8.5 | 10 mM | |
| NaCl | 500 mM | |
| EDTA | 1 mM |
- Gu6:
| A | B | |
| Guanidinium hydrochloride | 6 M | |
| Sodium phosphate, pH 8.5 | 20 mM |
- AE:
| A | B | |
| Ammonium acetate | 20 mM | |
| EDTA, pH 8.5 | 0.2 mM |
pET-Sac-Abeta(M1-42)addgeneCatalog #71875
Transformation and Aβ (M1-42) Expression
Transformation and Aβ (M1-42) Expression
Thaw RbCl-competent Escherichia coli Bl21 cells (DE3) On ice .
Add 1 µL of pET-Sac-Abeta(M1-42) plasmid (Addgene plasmid # 71875) 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.
Prepare preculture:
Pick 5 colonies and inoculate 13 mL LB/Ampicillin.
Shake at 37 °C for 4-6 h.
Take one clone and inoculate 200 mL LB/Ampicillin, shake Overnight at 37 °C .
Measure a 1:10 dilution of the overnight culture and inoculate 6 L of LB medium to an OD600 = 0.05.
Shake flasks at 90 rpm, 37°C until approx. OD600 = 0.4 (2.5 h).
Add isopropyl β-D-1-thiogalactopyranoside (IPTG) at final concentration of 0.4 millimolar (mM) .
Shake flasks at 80 rpm, 37°C, 04:00:00 (OD600 = 1.3).
Centrifuge the bacterial culture at 4000 rpm, 4°C, 00:30:00 . Discard supernatant.
30m
Resuspend each Liter of main culture with 20 mL cold double-distilled water and pipet in 50 mL tubes.
Centrifuge again at 5000 x g, 4°C, 00:30:00 , remove supernatant and flash-freeze in liquid nitrogen for storage at -80 °C .
30m
Lysis
Lysis
Thaw the cell pellets at Room temperature and resuspend them in 80 mL of TE 7.5 buffer supplemented with Complete EDTA-free protease inhibitor cocktail (Merck) and Sm DNase 50 µL .
Sonicate lysate On ice , 6 cycles 00:00:30 ON, 00:01:30 OFF (output 8, 53%).
Centrifuge lysate at 18000 x g, 4°C, 00:10:00 .
10m
Remove the supernatant (S1).
Resuspend the pellet in 80 mL of TE 7.5 buffer supplemented with Complete EDTA-free protease inhibitor cocktail (Merck) and Sm DNase 50 µL , until the sample is homogenous.
Sonicate lysate On ice , 6 cycles 00:00:30 ON, 00:01:30 OFF (output 8, 53%).
Centrifuge lysate at 18000 x g, 4°C, 00:10:00 .
10m
Remove the supernatant (S2).
Resuspend the pellet in 80 mL of TE 7.5 buffer until the sample is homogenous.
Sonicate lysate On ice , 6 cycles 00:00:30 ON, 00:01:30 OFF (output 8, 53%).
Centrifuge lysate at 18000 x g, 4°C, 00:10:00 .
10m
Remove the supernatant (S3).
Resuspend the pellet (inclusion bodies) using a glass rod and a magnetic stirrer with 80 mL of TE 9.5 buffer until the sample is homogenous.
Sonicate lysate On ice , 6 cycles 00:00:30 ON, 00:01:30 OFF (output 8, 53%).
Centrifuge lysate at 18000 x g, 4°C, 00:10:00 .
10m
Collect the supernatant in a glass beaker. If the sample is turbid, filter through a 0.22 µm filter.
Adjust the pH to 8.5 with 1 Molarity (M) HCl (around 6 drops).
Diethylaminoethyl Cellulose (DEAE-C) Chromatography (Anion Exchange)
Diethylaminoethyl Cellulose (DEAE-C) Chromatography (Anion Exchange)
Equilibrate the DEAE-C column (Cytiva, GE17-0709-10) with 2 column volumes (CV, 30 mL) TE 8.5 buffer.
Load supernatant from inclusion bodies resuspension to the DEAE-C column.
Wash the DEAE-C column with 4 CV TE 8.5 buffer.
Elute Aβ (M1-42) with 4 CV 10% TENa500 buffer and collect elution fractions.
Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.
DEAE-C chromatogram and SDS PAGE analysis. 5 µL sample + 5 µL 2x SDS sample loaded on a
4-20% SDS-PAGE.
Desalting and Lyophilization
Desalting and Lyophilization
Pool fractions containing Aβ (M1-42) (50-59).
In order to reduce the salt concentration, pass the eluted peptide through a HiPrep 26/10 desalting column equilibrated with the TE 8.5 buffer.
Pool the fraction containing Aβ (M1-42), and transfer the pool into a round bottom flask and freeze the sample while rotating in liquid nitrogen to cover the entire surface with ice.
Lyophilize the frozen sample.
Size Exclusion Chromatography
Size Exclusion Chromatography
Dissolve lyophilized Aβ (M1-42) to a final volume of 15 mL with Gu6 buffer.
Load the sample onto a Cytiva HiLoad 26/600 Superdex 75 pg SEC column previously equilibrated with AE buffer. Use AE as running buffer.
Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
Size exclusion chromatogram and SDS PAGE analysis. 5 µL sample + 5 µL 2x SDS sample
dye loaded on a 4-20% SDS-PAGE.
Pool fractions containing Aβ (M1-42) and measure peptide concentration.
Note
NOTE: Aβ (M1-42) contains just one tyrosine and no tryptophan, therefore measure peptide concentration in a Nanodrop at absorbance 205 nm (A205).
Aliquot the peptide in 500 µg aliquots in 1.5 mL tubes and flash-freeze in liquid nitrogen.
Lyophilize the sample, seal each tube in plastic foil to avoid rehydration and storage at -80 °C .
Protocol references
Linse S. Expression and Purification of Intrinsically Disordered Aβ Peptide and Setup of
Reproducible Aggregation Kinetics Experiment. Methods Mol Biol. 2020;2141:731-754. doi:
10.1007/978-1-0716-0524-0_38. PMID: 32696387.