Sauer:SspB purification

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 (amp^R, T7 promoter) and transformed NEB strain ER2566 with it. 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 (the same as the wash buffer above with the addition of 5 mM β-ME and 0.05% Tween-20) and frozen at -80 degrees.