Oct 28, 2021
Purification protocol of Mouse (Mus Musculus) E1-like enzyme ATG7
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Dorotea Fracchiolla 2021. Purification protocol of Mouse (Mus Musculus) E1-like enzyme ATG7. protocols.io https://dx.doi.org/10.17504/protocols.io.bsennbde
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: February 15, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 47278
Keywords: expression, purification, E1-like enzyme ATG7, autophagy, ASAPCRN
Abstract
This protocol outlines the procedures for expression and purification of Mouse (Mus Musculus) E1-like enzyme ATG7 (AuTophaGy-related protein) of the ATG8 ubiquitin-like conjugation system in autophagy.
Attachments
Image Attribution
© Dorotea Fracchiolla
Guidelines
General information: expression system: Sf9 cells, medium: SF921+Pen/Strep; plasmid origin: Gammoh Noor (University of Edinburgh); internal lab Construct Database number: SMC911; backbone: pFast-BacHT(B); resistance: Ampicillin/Gentamicin; insert: Mus musculus ATG7, Isoform 2_NP_00124647.1; tags & cleavage sites: N-term 6xHis, followed by Tobacco Etch Virus (TEV) cleavage site, 6xHis-TEVcs-mATG7; protein length without tags: 698 aa; Epsilon (all Cysteines reduced, with tags) = 88810 M-1 cm-1 ; MW with tags= 83 kDa; ORF (only mATG7):
MGDPGLAKLQFAPFNSALDVGFWHELTQKKLNEYRLDEAPKDIKGYYYNGDSAGLPTRLTLEFSAFDMSA STPAHCCPAMGTLHNTNTLEAFKTADKKLLLEQSANEIWEAIKSGAALENPMLLNKFLLLTFADLKKYHF YYWFCCPALCLPESIPLIRGPVSLDQRLSPKQIQALEHAYDDLCRAEGVTALPYFLFKYDDDTVLVSLLK HYSDFFQGQRTKITVGVYDPCNLAQYPGWPLRNFLVLAAHRWSGSFQSVEVLCFRDRTMQGARDVTHSII FEVKLPEMAFSPDCPKAVGWEKNQKGGMGPRMVNLSGCMDPKRLAESSVDLNLKLMCWRLVPTLDLDKVV SVKCLLLGAGTLGCNVARTLMGWGVRHVTFVDNAKISYSNPVRQPLYEFEDCLGGGKPKALAAAERLQKI FPGVNARGFNMSIPMPGHPVNFSDVTMEQARRDVEQLEQLIDNHDVIFLLMDTRESRWLPTVIAASKRKL VINAALGFDTFVVMRHGLKKPKQQGAGDLCPSHLVAPADLGSSLFANIPGYKLGCYFCNDVVAPGDSTRD RTLDQQCTVSRPGLAVIAGALAVELMVSVLQHPEGGYAIASSSDDRMNEPPTSLGLVPHQIRGFLSRFDN VLPVSLAFDKCTACSPKVLDQYEREGFTFLAKVFNSSHSFLEDLTGLTLLHQETQAAEIWDMSDEETV
Chromatograph and Coomassie BB stained gel of His-tag affinity purification.
Chromatograph and Coomassie BB stained gel of Size Exclusion purification.
Summary gel of mATG7 purification.
Materials
Materials and Reagents
- Sf9 insect cells
- SF921 medium with antibiotics 100 IU/ml Penicillin and 100 µg/ml Streptomycin
- sterile cell culture hood
- 27°C shaker incubator
- sterile flasks/sterile pipettes
- Baculovirus for mATG7 (SMC911)
- douncer 40 mL
Materials for Protein Purification
- Lysis Buffer: 50mM Hepes pH=7.5; 300mM NaCl, Benzonase (1μl/50ml lysis buffer), 10mM Imidazole, 2mM MgCl2, 1mM Dithiothreitol, 1xProtease Inhibitors/50 ml lysis buffer (EDTA-free CIP tablet, Roche).
- 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
- SEC Buffer: 25mM Hepes pH=7.5, 150mM NaCl (filtered and degassed) + 1mM 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)
- S200_16/60 (GE Healthcare)
Gels: 10% Poly-acrylamide SDS-gels
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Infection/expression/harvest
Infection/expression/harvest
Infect 1 L culture of Sf9 cells growing in Sf921 medium with Penicillin/Streptomycin at 1-1.5 mil/ml cells/volume at 99-100% viability in log phase with Virus 1 (V1) according to viral titer.
(NOTE: protein yield from 1 lt culture is high, e.i. 32 mg/1lt culture).
Monitor infection and harvest cells when viability goes to 97-98%.
Note
Always check under microscope: when all alive cells are brightly fluorescent and only few dead -> harvest!
Protein Purification
Protein Purification
45m
45m
Note
This section describes the procedure for His-Trap affinity purification followed by Size Exclusion Chromatography. All steps are to be executed at 4°C or on ice.
Resuspend cell pellet corresponding to 1 L culture in 50 mL ice cold lysis buffer ; gently stir at 4 °C until pellet dissolves avoiding bubbling.
Additionally, mechanically lyse the cells passing them through a pre-cooled douncer for 3x (10x pestle A followed by 10x pestle B).
Clear the lysate by spinning it in a Backman centrifuge, at 25K using a Ti45 Rotor for 00:45:00 at 4 °C .
45m
Inject the supernatant onto a 5ml HT column pre-equilibrated with Buffer A at 1ml/min flow rate to allow protein binding.
Wash the column for 5CV with Buffer A at 2 ml/min flow rate to remove unspecific bound proteins.
Elute protein through a step elution gradient in 50mM, 75mM, 100mM, 150mM, 200mM and 300mM Imidazole concentration. Perform the elution at 1ml/min flow rate.
Collect a sample from peak fractions of each elution step and check them on a SDS-PAGE (see gel in the "Guidelines" section). Pool and concentrate fractions corresponding to the peak containing the protein of interest (usually 200mM and/or 300mM Imid.) by spinning them down at4 °C using a 30 kDa cut-off Amicon Filter to 2 ml in a 5810R centrifuge (Eppendorf).
Note
Keep centrifugation steps short (00:05:00 ) to avoid protein local concentration/aggregation on the filter.
Inject 2 mL protein onto a S200_16/60 column at 44 °C pre-equilibrated in buffer containing 25mM Hepes pH=7.5, 150mM NaCl and 1mM DTT (see profile in the "Guidelines" section).
Check fractions on a 10% SDS-PAGE (see gel in the "Guidelines" section), pool and concentrate down those containing the protein of interest by spinning them at 4 °C using a 30kDa cut-off Amicon Filter in a 5810R centrifuge (Eppendorf). Protein elutes at around 72.5ml, a retention volume corresponding to that of a dimeric globular protein of ~150-160 kDa.
Note
Keep centrifugation steps short to avoid protein precipitation.
Measure protein absorbance at A280 is measured with Spectrophotometer against Size Exclusion Chromatography buffer.
Aliquot the protein, snap freeze it in liquid Nitrogen and store it at -80 °C .
Note
Usually protein activity is kept for 18 months when stored at -80°C.