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Contents

Background

I purified SspB using Igor's paper as a guide.

Expression

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.

Image:SspB 1.jpg Coomassie-stained SDS-PAGE showing SspB overexpression.

Buffers

I left the glycerol and magnesium out of the buffers.

Protocol

Lysis

Ammonium Sulfate Precipitation


Anion Exchange

Another Ammonium Sulfate Step

SEC

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):


Concentrate

Notes