| The expression plasmid pJC25NMstop was a gift from
Dr. Susan L. Lindquist (Whitehead Institute, Massachusetts
Institute of Technology). Sup35NM was purified as described
(Chernoff et al. 2002) with the following modification. Transformed
colonies were inoculated into a 50-mL LB-ampicillin
(50 mg/mL) flask and incubated for 5 h at 37 C, and then 20
mL of the growing culture was removed and added into a fresh
500-mL LB-Amp (50 mg/mL) medium. After induction with 1
mM IPTG at an A600 of 0.6, cells were harvested after 3 h of
induction. Cell pellets were collected by centrifugation and
stored at 80 C. Cells were lysed in 50 mL lysis buffer (10
mM Tris-HCl [pH 7.2], 1 mM DTT, 1 mM PMSF, 8 M urea)
for 30 min at 25 C. The lysate was then cleared by centrifugation
at 12,000g for 20 min at 10 C. The cleared supernatant
was applied to a Q Sepharose column (2.5 7.5 cm) pre-equilibrated
with lysis buffer. The column was washed with 5 bed
volumes of Q wash buffer I (10 mM Tris-HCl [pH 7.2], 1 mM
DTT, 1 mM PMSF, 8 M urea, 85 mM NaCl), and 5 volumes
of Q wash buffer II (10 mM Tris-HCl [pH 7.2], 8 M urea, 150
mM NaCl). The protein was eluted in 3 volumes of Q elution
buffer (10 mM Tris-HCl [pH 7.2], 8 M urea, 200 mM NaCl).
The eluant from the Q Sepharose was then loaded directly onto
a hydroxyapatite column (1.5 12 cm). The column was preequilibrated
with Q elution buffer. After loading, the column
was washed with 2 volumes of HA wash buffer I (1 mM
potassium phosphate [pH 6.8], 8 M urea, 1 M NaCl) and
then with 2 volumes of HA wash buffer II (25 mM potassium
phosphate [pH 6.8], 8 M urea). The protein was eluted using a
stepwise gradient of 75 mM and 125 mM potassium phosphate
(pH 6.8) in 8 M urea and then with 2 volumes of 125 mM
potassium phosphate (pH 6.8) in 8 M urea. Fractions (5 mL)
were analyzed by 15% SDS-PAGE (loading 10 mL per lane).
Protein concentrations were determined with the calculated
extinction coefficient of 0.90 for a 1 mg/mL Sup35NM solution
at 280 nm absorbance (Gill and von Hippel 1989). Anhydrous
methanol (100%) was added to precipitate Sup35NM on ice at
a ratio of 5:1. The supernatant was removed and the pellet was
stored in 70% (v/v) methanol (1/2 volume of supernatant) at
80 C (Serio et al. 1999). |