Feb 06, 2024
Cloning, Protein Expression, and Purification of 20S CPs and Assembly Intermediates
- 1Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
Protocol Citation: Frank Adolf 2024. Cloning, Protein Expression, and Purification of 20S CPs and Assembly Intermediates. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmd59l5b/v1
Manuscript citation:
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 05, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94683
Keywords: ASAPCRN, proteasome, core particle, 20S proteasome, chaperone, molecular machine, multiprotein complex, POMP, PAC1, PAC2, PAC3, PAC4, propeptide, protease
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP‐000282
Abstract
This protocol details methods for cloning, expression, and purification of 20S CPs and assembly intermediates for biochemical and structural analysis.
Guidelines
Please familiarise yourself with the laboratory safety rules and guidelines and follow these while performing the experiment. Please wear appropriate PE while performing the experiment.
Materials
X-tremeGENETM HP DNA Transfection Reagent - ROCHE
cOmplete PROTEASE INHIBITOR COKTAIL - ROCHE
Strep-Tactin‱ Sepharose‱ resin - IBA
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards. Liquid nitrogen (LN2) and other cryogens can cause severe damage to the skin and eyes. Always wear personal protective equipment when handling these cryogens.
Cloning of baculovirus transfer vectors
Cloning of baculovirus transfer vectors
1w
1w
Baculovirus transfer vectors were assembled utilizing a combination of the biGBac and MultiBac systems
CITATION
CITATION
The MultiBac vector pACEBac1 was used as both library vector and acceptor vector for step 1 biGBac assembly reactions with the following primers:
ACEBac-1EC-BBA.rev
ATTTAAATCTTTAGACCATAGAGCGTTCTCGCGAATCGATACTAGTGTTTAAACTCGCTACCTTAGGACC
ACEBac-2EC-BBA.fwd
ATTTAAATAAACCTAATGATGCCTGATGTTTCCTAGGGTATACCCATCTAATTGGAACCAGATAAGTGAAATC
ACEBac-3EC-BBA.fwd
ATTTAAATAAACGGTTCACATAGCTTAGTTTCCTAGGGTATACCCATCTAATTGGAACCAGATAAGTGAAATC
ACEBac-4EC-BBA.fwd
ATTTAAATAAACACTGACATTGACTTGGTTTCCTAGGGTATACCCATCTAATTGGAACCAGATAAGTGAAATC
ACEBac-5EC-BBA.fwd
ATTTAAATAAATCTATATCTCAATCGGGGTTCCTAGGGTATACCCATCTAATTGGAACCAGATAAGTGAAATC
Clone all 20S CP subunits and assembly chaperones into pACEBac1 using Gibson assembly
For affinity purification add C-terminal TEV-cleavable twin strep tags on β2 (PSMB7) and β7 (PSMB4)
1d
Screen for positive clones and sequence verify by Sanger sequencing
1d
Preparation of bigBac assembly inserts:
Amplify expression cassette from library vectors (step 2) by PCR with the following primers:
Cas1-ACEBac.fwd
AACGCTCTATGGTCTAAAGATTTAAATCGACCTACTCCGGAATATTAATAGATCATGG
Cas2-ACEBac.fwd
AAACTGGATACTATTGCACGTTTAAATCGACCTACTCCGGAATATTAATAGATCATGG
Cas3-ACEBac.fwd
AAACCTAATGATGCCTGATGTTTAAATCGACCTACTCCGGAATATTAATAGATCATGG
Cas4-ACEBac.fwd
AAACGGTTCACATAGCTTAGTTTAAATCGACCTACTCCGGAATATTAATAGATCATGG
Cas5-ACEBac.fwd
AAACACTGACATTGACTTGGTTTAAATCGACCTACTCCGGAATATTAATAGATCATGG
Cas1-ACEBac.rev
AAACGTGCAATAGTATCCAGTTTATTTAAATGGTTATGATAGTTATTGCTCAGCGGTGG
Cas2-ACEBac.rev
AAACATCAGGCATCATTAGGTTTATTTAAATGGTTATGATAGTTATTGCTCAGCGGTGG
Cas3-ACEBac.rev
AAACTAAGCTATGTGAACCGTTTATTTAAATGGTTATGATAGTTATTGCTCAGCGGTGG
Cas4-ACEBac.rev
AAACCAAGTCAATGTCAGTGTTTATTTAAATGGTTATGATAGTTATTGCTCAGCGGTGG
Cas5-ACEBac.rev
AACCCCGATTGAGATATAGATTTATTTAAATGGTTATGATAGTTATTGCTCAGCGGTGG
6h
Purify all inserts by agarose gel extraction
2h
Preparation of biGBac step 1 assembly acceptor vectors:
linearize the acceptor vector pACEBac1 by PCR with the following primers:
for cloning of 5 expression cassettes:
ACEBac-1EC-BBA.rev and ACEBac-5EC-BBA.fwd
for cloning of 4 expression cassettes
ACEBac-1EC-BBA.rev and ACEBac-4EC-BBA.fwd
for cloning of 3 expression cassettes
ACEBac1-1EC-BBA.rev and ACEBac-3EC-BBA.fwd
6h
Purify all linearized vectors by agarose gel extraction
2h
Clone multi expression cassette Baculo transfer vectors listed below by biGBac step 1 assembly with expression cassette inserts from step 3 and acceptor vectors from step 4
pACEBac1-POMP-PSMG1-PSMG2-PSMG3-PSMG4
pACEBac1-PSMA1-PSMA2-PSMA3-PSMA4,
pACEBac1-PSMA5-PSMA6-PSMA7,
pACEBac1-PSMB1-PSMB2-PSMB3-PSMB4,
pACEBac1-PSMB1-PSMB2-PSMB3-PSMB4-TEV-2xSTII,
pACEBac1-PSMB5-PSMB6-PSMB7,
pACEBac1-PSMB5-PSMB6-PSMB7-TEV-2xSTII.
1d
Screen for positive clones and sequence verify by Sanger sequencing
1d
Assemble finale multi expression cassette baculo transfer vectors by multibac assembly
pACEBac1-PSMA1-PSMA2-PSMA3-PSMA4-PSMA5-PSMA6-PSMA7
pACEBac1-PSMB1-PSMB2-PSMB3-PSMB4-TEV-2xSTII-PSMB5-PSMB6-PSMB7
pACEBac1-PSMB1-PSMB2-PSMB3-PSMB4-PSMB5-PSMB6-PSMB7-TEV-2xSTII
2d
Digest aceptor vectors listed below with I-CeuI, CIP and purify by agarose gel extraction
pACEBac1-PSMA5-PSMA6-PSMA7
pACEBac1-PSMB5-PSMB6-PSMB7
pACEBac1-PSMB5-PSMB6-PSMB7-TEV-2xSTII
4h
Digest donor vector listed below with I-CeuI and BstXI, and purify inserts by agarose gel extraction
pACEBac1-PSMA1-PSMA2-PSMA3-PSMA4
pACEBac1-PSMB1-PSMB2-PSMB3-PSMB4-TEV-2xSTII
pACEBac1-PSMB1-PSMB2-PSMB3-PSMB4
4h
Ligate the following vector/insert pairs listed below and transform in DH5alpha E. coli
pACEBac1-PSMA5-PSMA6-PSMA7 and EC1-4 PSMA1-PSMA2-PSMA3-PSMA4
pACEBac1-PSMB5-PSMB6-PSMB7 and EC1-4 PSMB1-PSMB2-PSMB3-PSMB4-TEV-2xSTII
pACEBac1-PSMB5-PSMB6-PSMB7 TEV-2xSTII and EC1-4 PSMB1-PSMB2-PSMB3-PSMB4
1d
Screen for positive clones and sequence verify by Sanger sequencing
1d
Baculo virus amplification and insect cell expression
Baculo virus amplification and insect cell expression
1w
1w
Culture Sf9 insect cells (Thermo Fisher Scientific) for virus amplification in serum-free Ex-cell 420 medium (Sigma-Aldrich)
Culture Trichoplusnia ni (Thermo Fisher Scientific) in protein free ESF 921 insect cell culture media (Expression Systems LLC)
All steps were carried out according to standard protocols
CITATION
LINK
https://doi.org/CITATION
For bacmid preparation transform baculo transfer vector from the section above into EMBACY E. coli and plate on LB-agar plates with ampicillin, kanamycin, tetracyclin, gentamycin, IPTG, and XGal
1d
Prepare Overnight cultures from white colonies in LB with ampicillin, kanamycin, and gentamycin
1d
Prepare bacmids by alkaline lysis and subsequent isopropanol and 70 % (v/v) EtOH precipitation
2h
Air dry bacmid pellets, resuspend DNA in milliQ water, and store at 4 °C until usage
1h
P1 virus production
2d 12h
For P1 viruis production seed 0.7 - 0.8x106 cells/well in 3 mL medium in a six well plate and leave for 00:15:00 min at 27 °C
15m
Prepare bacmids by diluting 1 µg bacmid DNA in 20 µL milliQ water, and add 200 µL of medium
5m
For each bacmid mix 100 µL medium with 15 µL Extreme Gene HP DNA transfection reagent (Roche)
5m
Add 112 µL of Medium Extreme Gene Mix to each bacmid DNA
5m
Add 156 µL of the DNA-Extreme Gene Mix to each well in the six-well plate
5m
Wrap the plates with Parafilm and incubate for 60:00:00 h at 27 °C
2d 12h
Transfer medium containing P1 virus from each well into a 15 mL tube and store at 4 °C until further usage
10m
P2 and P3 virus amplification
2d
Seed 0.8 - 1.0 x 106 Sf9 cells/ml into an appropriate conical flask and infect cells with 0.5 - 1.0% P1 or P2, respectively
1h
Incubate cell suspensions at 27 °C at 80 rpm for 48:00:00 h
2d
Harvest P2 and P3 by centrifugation, transfer medium containing virus into a 50 mL tube or 250 mL bottle and store at 4 °C until further usage
15m
Protein Expression in High FiveTM insect cells
2d 12h
Seed 2.0 x 106 High FiveTM cells/ml into an appropriate conical flask and infect with 0.5 - 1.0 % P3 virus
30m
Incubate cell suspensions at 27 °C at 80 rpm for 60:00:00 h
2d 12h
Harvest High FiveTM cells by centrifugation, resuspend cell pellets in PBS, transfer to a 50 mL tube, harvest cells by centrifugation, discard supernatant, snap freeze cell pellets in LN2, and store util further usage at -80 °C
30m
Purification of 20S CPs and 20S CP assembly intermediates
Purification of 20S CPs and 20S CP assembly intermediates
8h
8h
Purification of mature 20S CPs together with their assembly intermediates
8h
Thaw cell pellets form section 2 - step 11, resuspend in buffer A 25 mM HEPES pH 7.5 (KOH), 150 millimolar (mM) NaCl, 1 millimolar (mM) DTT, and add 1 tablet cOmplete protease inhibitor mix (Merck) per 10 mL cell pellet resuspended in 40 mL buffer A in a 50 ml tube
30m
Lyse cells by sonication on ice
10m
Centrifuge lysate at 22k at 4 °C for 01:00:00 h
1h
In parallel wash Strep-Tactin‱ Sepharose‱ resin (IBA) 3 times with buffer A
45m
Incubate supernatants from step 12.3 with resin for 01:00:00 h at 4 °C
1h
Wash resin 3 times with buffer A by centrifugation at 2000 x g for 00:15:00 min at 4 °C
15m
Load resin on a gravity flow column and elute proteins with 2.5 micromolar (µM) d-Desthiobiotin in buffer A
15m
Pool fractions of interest, concentrate in a spin protein concentrator with a MWCO of 100 kDa to max 5-8 mg/mL
1h
In parallel equilibrate a Superose 6 10/300 GL column (Cytiva) with buffer A
1h
Apply concentrated eluate from step 12.8 onto the SEC column at a flow rate of 1 ml/min, record absorption at 280 nm, and fractionate elution a 300 µL
1h
Analyze SEC fractions by SDS-PAGE, pool fractions of interest, snap freeze in LN2, and store util further usage at -80 °C for biochemical assays
For structural analysis use fractions directly after SEC
2h
Citations
Step 7
Fitzgerald DJ, Berger P, Schaffitzel C, Yamada K, Richmond TJ, Berger I. Protein complex expression by using multigene baculoviral vectors.
https://doi.org/Step 7
Bieniossek C, Richmond TJ, Berger I. MultiBac: multigene baculovirus-based eukaryotic protein complex production.
https://doi.org/10.1002/0471140864.ps0520s51