Cloning, Protein Expression, and Purification of 20S CPs and Assembly Intermediates

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

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)

Screen for positive clones and sequence verify by Sanger sequencing  

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

Purify all inserts by agarose gel extraction

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

Purify all linearized vectors by agarose gel extraction

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.

Screen for positive clones and sequence verify by Sanger sequencing

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

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

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

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

Screen for positive clones and sequence verify by Sanger sequencing

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

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

Prepare cultures from white colonies in LB with ampicillin, kanamycin, and gentamycin

Prepare bacmids by alkaline lysis and subsequent isopropanol and 70% (v/v) EtOH precipitation

Air dry bacmid pellets, resuspend DNA in milliQ water, and store at 4°C until usage

P1 virus production

For P1 viruis production seed 0.7 - 0.8x10⁶ cells/well in 3mL medium in a six well plate and leave for 0h 15m 0s min at 27°C

Prepare bacmids by diluting 1µg bacmid DNA in 20µL milliQ water, and add 200µL of medium

For each bacmid mix 100µL medium with 15µL Extreme Gene HP DNA transfection reagent (Roche)

Add 112µL of Medium Extreme Gene Mix to each bacmid DNA

Add 156µL of the DNA-Extreme Gene Mix to each well in the six-well plate

Wrap the plates with Parafilm and incubate for 60h 0m 0s h at 27°C

Transfer medium containing P1 virus from each well into a 15mL tube and store at 4°C until further usage

P2 and P3 virus amplification

Seed 0.8 - 1.0 x 10⁶Sf9 cells/ml into an appropriate conical flask and infect cells with 0.5 - 1.0% P1 or P2, respectively

Incubate cell suspensions at 27°C at 80 rpm for 48h 0m 0s h

Harvest P2 and P3 by centrifugation, transfer medium containing virus into a 50mL tube or 250mL bottle and store at 4°C until further usage

Protein Expression in High FiveTM insect cells

Seed 2.0 x 10⁶ High FiveTM cells/ml into an appropriate conical flask and infect with 0.5 - 1.0 % P3 virus

Incubate cell suspensions at 27°C at 80 rpm for 60h 0m 0s h

Harvest High FiveTM cells by centrifugation, resuspend cell pellets in PBS, transfer to a 50mL tube, harvest cells by centrifugation, discard supernatant, snap freeze cell pellets in LN2, and store util further usage at -80°C

Purification of mature 20S CPs together with their assembly intermediates

Thaw cell pellets form section 2 - step 11, resuspend in buffer A 25 mM HEPES pH 7.5 (KOH), 150millimolar (mM) NaCl, 1millimolar (mM) DTT, and add 1 tablet cOmplete protease inhibitor mix (Merck) per 10mL cell pellet resuspended in 40mL buffer A in a 50 ml tube

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 300µL

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

Lyse cells by sonication on ice

Centrifuge lysate at 22k at 4°C for 1h 0m 0s h

In parallel wash Strep-Tactin^® Sepharose^® resin (IBA) 3 times with buffer A

Incubate supernatants from step 12.3 with resin for 1h 0m 0s h at 4°C

Wash resin 3 times with buffer A by centrifugation at 2000 x g for 0h 15m 0s min at 4°C

Load resin on a gravity flow column and elute proteins with 2.5micromolar (µM) d -Desthiobiotin in buffer A

Pool fractions of interest, concentrate in a spin protein concentrator with a MWCO of 100 kDa to max 5-8mg/mL

In parallel equilibrate a Superose 6 10/300 GL column (Cytiva) with buffer A