TITLE: Cloning, Protein Expression, and Purification of 20S CPs and  Assembly Intermediates
AUTHORS: Frank Adolf

[DESCRIPTION]
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.

[STEPS]
SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12. Purification of mature 20S CPs together with their assembly intermediates
SECTION: Baculo virus amplification and insect cell expression
7. 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


 

 

SECTION: Cloning of baculovirus transfer vectors
1. Baculovirus transfer vectors were assembled utilizing a combination of the biGBac and  MultiBac systems

 

 

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
SECTION: Cloning of baculovirus transfer vectors
2. 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)
SECTION: Cloning of baculovirus transfer vectors
3. 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
SECTION: Cloning of baculovirus transfer vectors
4. 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
SECTION: Cloning of baculovirus transfer vectors
5. 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. 
SECTION: Cloning of baculovirus transfer vectors
3.1. Purify all inserts by agarose gel extraction
SECTION: Cloning of baculovirus transfer vectors
6. 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
SECTION: Cloning of baculovirus transfer vectors
4.1. Purify all linearized vectors by agarose gel extraction
SECTION: Cloning of baculovirus transfer vectors
5.1. Screen for positive clones and sequence verify by Sanger sequencing 
SECTION: Cloning of baculovirus transfer vectors
2.1. Screen for positive clones and sequence verify by Sanger sequencing  
SECTION: Cloning of baculovirus transfer vectors
6.1. 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
SECTION: Cloning of baculovirus transfer vectors
6.2. 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
SECTION: Cloning of baculovirus transfer vectors
6.3. 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
SECTION: Cloning of baculovirus transfer vectors
6.4. Screen for positive clones and sequence verify by Sanger sequencing
SECTION: Baculo virus amplification and insect cell expression
8. 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
SECTION: Baculo virus amplification and insect cell expression
8.1. Prepare   cultures from white colonies in LB with ampicillin, kanamycin, and gentamycin

SECTION: Baculo virus amplification and insect cell expression
8.2. Prepare bacmids by alkaline lysis and subsequent isopropanol and 70 % (v/v) EtOH precipitation

SECTION: Baculo virus amplification and insect cell expression
8.3. Air dry bacmid pellets, resuspend DNA in milliQ water, and store at 4 °C until usage

SECTION: Baculo virus amplification and insect cell expression
9. P1 virus production

SECTION: Baculo virus amplification and insect cell expression
9.1. For P1 viruis production seed 0.7 - 0.8x106 cells/well in 3 mL medium in a six well plate and leave for 15 min min at 27 °C

SECTION: Baculo virus amplification and insect cell expression
9.2. Prepare bacmids by diluting 1 µg bacmid DNA in 20 µL milliQ water, and add 200 µL of medium

SECTION: Baculo virus amplification and insect cell expression
9.3. For each bacmid mix 100 µL medium with 15 µL Extreme Gene HP DNA transfection reagent (Roche)

SECTION: Baculo virus amplification and insect cell expression
9.4. Add 112 µL of Medium Extreme Gene Mix to each bacmid DNA

SECTION: Baculo virus amplification and insect cell expression
9.5. Add 156 µL of the DNA-Extreme Gene Mix to each well in the six-well plate

SECTION: Baculo virus amplification and insect cell expression
9.6. Wrap the plates with Parafilm and incubate for 3600 min h  at 27 °C

SECTION: Baculo virus amplification and insect cell expression
9.7. Transfer medium containing P1 virus from each well into a 15 mL tube and store at 4 °C until further usage 

SECTION: Baculo virus amplification and insect cell expression
10. P2 and P3 virus amplification 
SECTION: Baculo virus amplification and insect cell expression
10.1. 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  
SECTION: Baculo virus amplification and insect cell expression
10.2. Incubate cell suspensions at 27 °C at 80 rpm for 2880 min h

SECTION: Baculo virus amplification and insect cell expression
10.3. 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

SECTION: Baculo virus amplification and insect cell expression
11. Protein Expression in High FiveTM insect cells
SECTION: Baculo virus amplification and insect cell expression
11.1. Seed  2.0 x 106  High FiveTM cells/ml into an appropriate conical flask and infect with 0.5 - 1.0 % P3 virus
SECTION: Baculo virus amplification and insect cell expression
11.2. Incubate cell suspensions at 27 °C at 80 rpm for 3600 min h

SECTION: Baculo virus amplification and insect cell expression
11.3. 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

SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.1. 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

SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.2. Lyse cells by sonication on ice 
SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.3. Centrifuge lysate at 22k at 4 °C for 60 min h

SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.4. In parallel wash Strep-Tactin‱ Sepharose‱ resin (IBA) 3 times with buffer A 
SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.5. Incubate supernatants from step 12.3 with resin for 60 min h at 4 °C

SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.6. Wash resin 3 times with buffer A by centrifugation at 2000 x g for 15 min min at  4 °C

SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.7. Load resin on a gravity flow column and elute proteins with 2.5 micromolar (µM) d-Desthiobiotin in buffer A

SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.8. Pool fractions of interest, concentrate in a spin protein concentrator with a MWCO of 100 kDa to max 5-8 mg/mL

SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.9. In parallel equilibrate a Superose 6 10/300 GL column (Cytiva) with buffer A 
SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.10. 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 

SECTION: Purification of 20S CPs and 20S CP assembly intermediates
12.11. 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