PCquad and O3-33 Expression and Purification

Growth and Pelleting:

1. Inoculate 5 ml of cells in 37 C overnight
2. Incubate cells in larger flask(s) (1 - 4 L per batch) in 37 C until OD reaches 0.8
3. Add 0.5 mM IPTG (1 mL of 0.5 M stock per liter of cells)
4. Incubate cells in 30 C for 5 hrs
5. Aliquot cells into 50 mL Falcon tubes
6. Wash and pellet cells
1. Centrifuge cells 4000g for 10 min
2. Dump out supernatant
3. Resuspend cells in 10 ml of cold water
4. Consolidate suspended pellets into fewer falcon tubes
5. Centrifuge…    Repeat until all cells washed into a single tube

Lysis and filtration:

1. Resuspend cell pellet in lysis buffer (1 : 2    cell weight : ml of lysis buffer)
1. PCQuad: 20mM sodium phosphate (ph=8.0), 300 mM sodium chloride, 10 mM imidazole
2. O3-33: 50 mM TRIS (ph=8.0), 250 mM NaCl, 20mM imidazole
2. Chill cell suspension for 10 min
3. Add 10 uL 100 mM PMSF for every mL of cell suspension right before sonication
4. Sonicate cell suspension:for 10 sec (10 cycles; 30 sec pause between each cycle)
5. Centrifuge lysates for 30 min at 13000 rpm
6. Filter supernatant through a 22 um Millipore filter

Purification:

1. Used Hispur column purification protocol
1. Add appropriate amount of resin bed to purification column
2. Equilibriate column with two resin bed volumes of equilibration buffer
3. Add equal volume of equilibration buffer through filtered protein sample
4. Add sample through column. Collect flow through and reapply through column. Save flow through for downstream analysis if desired
5. Wash resin with two resin bed volumes of wash buffer with a gradient of imidazole concentrations until protein A280 reaches baseline Save flow through for downstream analysis if desired
6. Elute protein with two resin bed volumes of elution buffer. Repeat elution. Save flow through for downstream analysis if desired
7. Imidazole concentrations used:
1. PCQuad
1. Equilibration: 20 mM
2. W1: 25 mM, W2:40 mM, W3: 150 mM
3. E1: 400 mM, E2: 400 mM
2. O3-33
1. Equilibration: 20 mM
2. W1: 30 mM, W2: 40 mM, W3: 50 mM
3. E1: 400 mM, E2: 400 mM
2. Run SDS-PAGE on equilibration, three washes and two elutions
1. Mix samples with an equal volume of sample buffer (prepped with BME)
2. Heat samples at 70 C for 10 min before loading into gel
3. Use 1x SDS MOPS Running Buffer
3. Run Native-PAGE gel on best elutions
1. Use MOPS Running Buffer
4. Syringe filter 1 mL of best elutions through 0.22 um filter and use 30 uL for DLS

Buffers Preparation Protocols:

O3-33:

• Lysis buffer: 50 mM TRIS (ph=8.0), 250 mM NaCl, 20mM imidazole supplemented with 1 mM phenylmethanesulfonyl fluoride
• 50 ml solution
• 2.5 mL TRIS
• 0.7305 g NaCl
• 0.068 g imidazole
• 0.0087 g phenylmethanesulfonyl fluoride
• Up to 50 ml water

PCQuad:

• Lysis buffer: 20mM sodium phosphate (ph=8.0), 300 mM sodium chloride, 10 mM imidazole
• 50 ml solution
• .164 g sodium phosphate
• .877 g NaCl
• .034 g imidazole
• Up to 50 ml water
• Lysis buffer supplemented with 500 mM imidazole(Elution)
• 50 ml solution
• 1.702 g imidazole
• Up to 50 ml lysis buffer