I purified SspB using Igor's paper as a guide.
I obtained a clone of wild-type E. coli sspB in pET-3a (ampR, T7 promoter) from Igor and transformed NEB strain ER2566 with it. Don't ask Igor for the clone, he has given it many times to the Sauer lab. ER2566 is a BL-21 derivative that has the restriction systems knocked out. Like BL-21, ER2566 lacks the lon and ompT proteases. When growing cultures for stocks or larger overnights as starter cultures, I had 0.2% glucose in the medium to reduce lac promoter activity.
I started 2 x 1L cultures from a 1/100 dilution of an overnight culture. The LB was supplemented with 150 ug/mL ampicillin and 0.2% glycerol. At an OD of ~1.0, I added 1.0g lactose, 0.5g AmSO4 and 1 mM IPTG all in powder form. The expression went for 3 h at 37 degrees.
The culure was chilled, harvested, and resuspended in 25 mLs/L ice cold wash buffer (25 mM HEPES-Tris, 2 mM Mg-OAc, 0.5 mM CaCl2, 150 mM NaCl, pH 7.6), and transferred to two 50 mL Falcon tubes.
The cells were re-harvested and resuspended in 10 mLs lysis buffer per liter (the same as the wash buffer above with the addition of 5 mM β-ME and 0.05% Tween-20), then PMSF was added to 1 mM and the cells were frozen at -80 degrees.
I left the glycerol and magnesium out of the buffers.
- "5X" Buffer-7.6: 125 mM HEPES-Tris, 2.5 mM EDTA, pH 7.6. Start with 125 mM HEPES than slowly add Tris base until the pH is 7.6.
- 1M KCl made with 1/50th Buffer-7.6. Make sure the pH is near 7.6.
- "5X" Buffer-6: 125 mM K+-MES, 1 mM EDTA, pH 6.0.
- 1M NaCl made with 1/50th buffer 6. Check the pH to make sure it's near 6.
- When I prepared "1X" buffers, I added Tween-20 to 0.01%. Although SspB is fine without Tween, most of the plastic-ware people use (like microfuge tubes) binds proteins, the Tween stops this.
- The cells were thawed and lysozyme was added to ~0.25 mg/mL as a powder.
- Another shot of PMSF to 1 mM and mix well.
- After lysis (about 5 mins at room temp.) 3 μL of Benzonase was added.
- After the viscosity reduced noticeably, more lysis buffer was added to make each tube have 20 mLs and EDTA was added to 5 mM final.
- Transfer the lysates to centrifuge tubes and let them cool on ice for 10 minutes. The EDTA chelated the Mg++, so ribosomes and other metal-dependent complexes are destabilized, many of their proteins will want to precipitate, this step allows that.
- Clear the lysate. I spun for 15 minutes at 12K in the SA-600.
Ammonium Sulfate Precipitation
- Meanwhile, weigh out AmSO4 to make 20 mLs at 30% saturation (0.164 g/mL) and place the AmSO4 in clean centrifuge tubes.
- After the spin, decant the supernatant into the tubes with AmSO4 and rotate them at 4 degrees for 30 minutes. The will precipitate SspB. Fortunately, most other things are soluble.
- Centrifuge the samples to collect the SspB AmSO4 pellet (12K, 15 minutes).
- Decant the supernatant.
- Add ~30 mLs of "no salt" 1X Buffer-6. There is plenty of salt remaining in the AmSO4 pellet and it has to be diluted. I just filled the centifuge tubes with buffer.
- Re-solubilize SspB by rotating the tubes ~15 minutes at 4 degrees.
- Spin the tubes to clear the insoluble material.
- Transfer the sup. to a clean container, you should have about 60-80 mLs.
- While those spins are going on, prepare an anion-exchange column (usually some form of Q-column) by rinsing it with 1M NaCl (pH 6.0) and then with Buffer-6 with 50 mM NaCl (Line-A6 buffer). I used two Hi-Trap Q XP column screwed together. Prep. the FPLC with Line-A6 buffer containing 50 mM NaCl and Line-B6 buffer with 500 mM NaCl. If you plan to freeze the fractions from this step, you can add glycerol to each buffer to 10%. This helps proteins when they freeze, but I don't think SspB needs it.
- Load the column. I usually load by hand with a 60 mL syringe. You can use a peristaltic pump or a super-loop, whatever you like.
- After loading, allow Line-A6 buffer to flow over th column until a stable baseline is reached.
- Run a gradient to elute the SspB. It came off between 250 and 300 mM NaCl. You can step the column to 150 mM, then run a shallow gradient to elute. SspB should be a huge peak.
- I ran a gel of the peak fractions and found that most of the contaminants were toward the beginning of the SspB peak. I ditched them and pooled the majority of the SspB fractions and froze it at -80.
Another Ammonium Sulfate Step
- To concentrate for the SEC step, bring the thawed fractions from the anion-exchange column to 35% saturation again. You can precipitate the prtoein overnight at 4 degrees.
- (You might want to start preparing the SEC column now).
- After harvesting the SspB, drain the pellet well.
- Add ~1.5 mL of "no-salt" 1X Buffer-7.6 and gently shake the protien into solution. This should be enough to get the SspB into solution. Our injection loop for the Superdex-75 step was only 2 mLs, so I made an effort to keep the volume below that.
- Transfer the protein to a microfuge tube and spin it 10 minutes to clear aggregates. Then transfer to a clean tube on ice.
Pick a column that will include the SspB dimer. An S-100 or S-75 should be fine. Follow the manufacturer's instructions as far as maximum injection and pressure/flow rate.
Superdex-75 (16X60 mm):
- 0.3 MPa max pressure (set pressure limit).
- 1.0 mL/min flow rate (slower generally better, but there isn't too much difference).
- When removing the 20% ehtanol storage solution, first wash the column and pump with filtered water before swithing to your buffer.
- I ran SspB in Buffer-7.6 with 100 mM KCl, 0.01% Tween, 5 mM β-ME. (SspB doesn't have cysteins, but other aggregates might be.
- I collected 4 mL fractions.
- It eluted around a 45-50 kDa protein.
- Run a gel to check your peak fractions. The majority of mine was in three fractions. These were pooled.
- I concentrated SspB in a Millipore 10-K centrifugal concentrator. You could use Ammonium sulfate and buffer exchange. The concentration off of the sizing column was probably high enough without concentrating.
- I washed the protein to buffer exchange into a buffer I do my degradation reactions in.
- I rinsed the concentrator, pooled the protein, and gently mixed.
- I measured the concentration after dilution into 6M Guanidine-Cl, pH 7.0 (PO4). The extincion coefficient at 280 nm in Gu-Cl for the momoner is 12,090.
- My final stocks were ~1.1 mM.