General protocol for 10X-His-pET-Smt3-Gene-of-interest expression and purification:
1. Plasmid pET-His10Smt3-X-gene was transformed into Escherichia coli BL21(DE3).
2. Innoculate 10 ml LB-Kan (final 50 ug/ml of Kan) with 5-10 colonies and shake for 4
hours at 37°C. Transfer to 50 ml LB-Kan and shake for 2-3 hours at 37°C.
3. Transfer to 1L LB-Kan and shake to OD600 = 0.6-0.8 (takes 1-2 hours). Transfer flasks
to cold room and incubate for 1 hour. In the mean time adjust temp of incubator to 17°C.
4. Adjust cooled cultures to 2% ethanol and 1 mM IPTG respectively.
5. The cultures were then incubated at 17° C for 17 h with constant shaking (225 rpm).
6. Cells were harvested by centrifugation (3.5K rpm for 30 min) and the pellets stored at
-80°C. All subsequent procedures were performed at 4°C.
7. The cell pellets were resuspended in 50 mM Tris-HCl, pH 8.0, 350 mM NaCl, 10%
glycerol, 25 mM imidazole, 1 mM BME, 0.05% Triton-X100 (buffer A). PMSF, lysozyme,
and Triton-X100 were added to final concentrations of 0.2 mM, 5 mg/ml, and 0.2%
respectively and the cells allowed to lyse on ice for 30 min. The lysates were sonicated
(amplitude 9, total pulse time 2 min) to reduce viscosity and centrifuged (at 19K rpm for
90 min) to remove insoluble material.
8. The supernatant was applied to a 1-ml Ni2+-NTA-agarose column equilibrated with
buffer A. The column was washed with 5 ml of the same buffer followed by 5 ml of 60
mM imidazole in buffer A. The Smt3-fusion proteins were eluted with 350 mM imidazole
in buffer A.
9. The Smt3 tag was cleaved by adding Ulp1 (His10Smt3-X:Ulp1 ratio of 250:1) and
dialysis against 50 mM Tris(8.0), 350 mM NaCl, 40 mM Imidazole, 0.05% Triton-X100, 1
mM BME o/n at 4˚C. This material was (concentrated if necessary and) applied to a
second Ni-NTA column equilibrated with 40 mM imidazole in buffer A. The tag-free fulllength proteins are recovered in the flow through and wash fractions (2 column volumes
of 40 mM imidazole in buffer A) and concentrated for gel-filtration purposes.
9. The concentrated protein is applied to the Superdex75/200 column equilibrated with
20 mM Tris (8.0), 350 mM NaCl, 1 mM BME. The fractions are analyzed using SDSPAGE, concentrated, adjusted to 10% glycerol and frozen in 50-100 ul aliquots in liquid
N2.