Dec 02, 2022
Expression and purification protocol of the Human (Homo sapiens) LC3B Ubiquitin-like modifier
- 1Team Hurley;
- 2University of California, Berkeley
Protocol Citation: Dorotea Fracchiolla, Liv Jensen 2022. Expression and purification protocol of the Human (Homo sapiens) LC3B Ubiquitin-like modifier. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw82dl5r/v1
Manuscript citation:
https://doi.org/10.1083/jcb.201912098
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: December 02, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 73497
Keywords: expression, purification, recombinant, Microtubule-associated proteins 1A/1B light chain 3B, LC3B, mCherry-tag, ASAPCRN
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol describes expression and purification procedures for obtaining mCherry-tagged human recombinant Ubiquitin-like modifier LC3B (MAP1LC3B, Microtubule-associated proteins 1A/1B light chain 3B) lacking five C-terminal
amino acids to allow in vitro protein conjugation to target PE, Phosphatidyl Ethanolamine.
Image Attribution
© Dorotea Fracchiolla
Guidelines
Affinity purification
Chromatograph of His-tag affinity purification for mCherry-hLC3B.
Coomassie BB stained gel of His-tag affinity purification for mCherry-hLC3B.
Size Exclusion Chromatography
Chromatograph of Size Exclusion purification for mCherry-hLC3B.
Coomassie BB stained gel of Size Exclusion Chromatography purification for mCherry-hLC3B.
Materials
Materials and Reagents
- Escherichia coli Rosetta pLyss cells
- Luria Bertani (LB) medium with antibiotics (final conc. 50µg/ml Ampicillin, 34µg/ml Chloramphenicol)
- IPTG (isopropyl-b-d-thiogalactopyranoside)
- 37°C shaker incubator
- sterile flasks/sterile pipettes
- tip sonicator
- Lysis Buffer: 50mM Hepes pH=7.5, 300mM NaCl, 10mM Imidazole, 2mM MgCl2, 2mM β-mercaptoethanol, 1mM Pefablock, Complete Protease Inhibitors (EDTA-free CIP tablet, Roche), DNAse (Sigma).
- Buffer A: 50mM Hepes pH=7.5, 300mM NaCl, 10mM Imidazole (filtered and degassed) + 1mM β-mercaptoethanol;
- Buffer B: 50mM Hepes pH=7.5, 300mM NaCl, 300mM Imidazole (filtered and degassed) + 1mM β-mercaptoethanol;
- Size Exclusion Chromatography (SEC) Buffer: 25mM Hepes pH=7.5, 150mM NaCl, 1mM DTT (Dithiothreitol).
Note: all purification buffers are filtered and degassed. Reducing agents (β-mercaptoethanol and Dithiothreitol) are added after degassing step.
Columns:- HT 5ml column (GE Healthcare)
- S75_16/60 (GE Healthcare)
Gels:10% SDS-PAGE
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
General information
Insert: Homo sapiens LC3B, NP_073729.1; Expression system: E.Coli Rosetta pLyss; plasmid origin: Sascha Martens Lab, Addgene 169168, lab internal construct database number SMC948; backbone: pET-Duet1; plasmid resistance: Ampicillin; tags & cleavage sites: N-term 6xHis, followed by Tobacco Etch Virus (TEV) cleavage site, mCherry tag, LC3B ORF. Ext coeff: 41830 M-1 cm-1 , MW 47,89 kDa.
Protein Expression
Protein Expression
16h 15m
16h 15m
Transform plasmid DNA (Addgene_190237) into E.Coli Rosetta pLyss cells and plate on Ampicillin LB agar plate for Overnight at 37 °C .
The following day, inoculate a 5 mL LB + Amp/Cam pre-culture with 1-2 colonies and grow Overnight at 37 °C shaking.
The following day, use 5 mL pre-culture to inoculate 1 L LB medium + Amp/Cam at 37 °C until an OD600 (Optical Density) of 0.6 is reached.
Induce protein expression with 400 micromolar (µM) IPTG and grow for a further 16:00:00 at 18 °C shaking.
16h
Pellet cells at 4000 rpm, 4°C, 00:15:00 in a Sorvall RC6+ centrifuge (Thermo Scientific), discard supernatant and resuspend pellets in ice cold lysis buffer (25 ml/1 lt culture).
15m
Flash freeze resuspended pellets in liquid nitrogen and store at -80 °C until purification.
Protein Purification
Protein Purification
Perform His-Trap affinity purification followed by Size Exclusion Chromatography.
Purification of LC3B Ubiquitin-like modifier
Purification of LC3B Ubiquitin-like modifier
30m 30s
30m 30s
Cells are lised via freeze/thaw cycles and sonication: thaw pellet corresponding to 1 L culture by freeze/thawing in Room temperature water bath. All following steps are to be executed at 4 °C or On ice .
Lyse cells by sonicating them with an immersion Tip Sonicator (2x 00:00:30 ). Note: adjust times and intensity according to the available instrument.
30s
Clear lysate by spinning in a Beckman centrifuge, at 40000 x g, 4°C, 00:30:00 , Ti45 Rotor .
30m
Inject soluble fraction onto a 5ml HT column operating at 4 °C pre-equilibrated in Buffer A at 1ml/min flow rate.
Wash column for 5 undetermined with Buffer A at 2 ml/min flow rate to remove unspecific bound proteins.
Elute protein at 300mM Imidazole concentration. Perform elution at 1ml/min flow rate.
Perform TEV cleavage with 1mg/ml TEV protease overnight at 4˚C while dialyzing against elution buffer containing no imidazole.
Apply cleavage reaction to equilibrated HT column, and collect the flow-through.
Measure protein absorbance at A280 with a Spectrophotometer against dialysis buffer.
Aliquot protein, flash freeze in liquid Nitrogen and store at -80 °C .