| pRSET A?TcP0 was transferred to E. coli BL21-
(DE3)pLysS-competent cells (Invitrogen). Colonies from this transformation were grown at 378C up to an optical density of 0.6 at 600 nm. The cultures were induced with 0.1 mM isopropyl- b-D-thiogalactoside (IPTG) and grown for an additional 2 h at the same temperature. The bacteria were recovered by centrifugation, resuspended in native buffer (Na2HPO4 50 mM, NaCl 300 mM, pH 8), and lysed by freezing?defrosting cycles and sonication.
The lysate was centrifuged at 1000g for 20 min and the pellet was resuspended in 10 vol of native buffer, washed, and centrifuged twice. The final pellet was resuspended in 10 vol of buffer A (8 M urea, 10 mM Tris, 100 mM Na2HPO4, 10 mM2-mercaptoethanol, pH 8), left for 1 h at room temperature and finally centrifuged for 1 h at 10,000g. The supernatant was mixed with the Ni?NTA resin (Qiagen), which had been previously equilibrated with buffer A (6 mg or more of TcP0 was added for each ml of resin). The mixture was incubated with slow agitation for 1 h at room temperature and poured into the column. The flowthrough was collected and the column subsequently washed four times with 3 vol of buffer A followed by four washes
with 3 vol of buffer B (8 M urea, 10 mM Tris, 100
mM Na2HPO4, 10 mM 2-mercaptoethanol, pH 6.3). The protein was eluted sequentially with buffer C (8Murea, 10mMTris, 100mMNa2HPO4, 10mM2-mercaptoethanol, pH 5.9) and buffer D (8 M urea, 10 mM Tris, 100 mM Na2HPO4, 10 mM 2-mercaptoethanol, pH 4.5). Ten fractions (1 column vol per fraction) were collected with
each buffer. The eluted fractions were analyzed by 12% SDS?PAGE. The protein concentration was estimated by the method of Bradford (31) using bovine serum albumin as standard.
One volume of TcP0 in elution buffer (C or D) was fractionated in ten aliquots and added sequentially at intervals of 20 min, with stirring, to 50 vol of refolding buffer (0.3 M Tris?HCl, pH 8.8?.01% SDS (w/v)?0% glycerol (v/v)) at a temperature of 48C. The final protein concentration was 50 mg/ml. The sample was left overnight at 48C and then concentrated to the original volume by filtration using Centricon tubes (Amicon) with a 10-kDa cutoff. |