Linda Warfield, Hahn Lab, 2003
Volumes given are for 2L of cells for each subunit (Toa1 and Toa2).
Toa1 and Toa2 subunits are expressed separately in cultures of OD 0.6 for 2 hours with 0.4mM IPTG at 37 degrees. Cells are washed in RB buffer, pelleted, quickly frozen and stored at
-70 degrees.
Cells are thawed, resuspended in 60ml RB Buffer + 5mM DTT + protease inhibitors and disrupted by two passes through the microfluidizer. Insoluble material is pelleted for 10 minutes at 8.5K in GSA rotor at 4 degrees. Supernatant is discarded, pellets are washed in 100ml RB, and pelleted again. Supernatant is discarded and the pellets are resuspended in RB Buffer + 7M urea + 5mM DTT + PIs. This is incubated on a platform rotator for 1 hour at 4 degrees to resolubilize the subunits. Insoluble material is pelleted for 10 minutes at 10K in SS-34 rotor at 4 degrees. Save supernatant.
At this point, a gel can be run with induced cells, crude extract, and urea extract. Most of the protein in the urea extract should be the expressed TFIIA subunit. A protein assay is done, then the two subunits are mixed together in equimolar amounts at 0.4 �C 1 mg/ml (additional RB + 7M urea is added to obtain correct concentration for renaturation). The two subunits are then renatured together during dialysis against RB Buffer (no urea) + 5mM DTT + PIs with 3 changes of buffer over approximately 20 hours. Renatured protein is centrifuged for 6 minutes at 10K in SS-34 rotor.
After the renaturation step, I either concentrate the protein on a Centriprep-10 concentrator (I’ve been told that other concentrators tend to give a lower yield) and then add no salt buffer so the final salt is 50mM KCl. Or I just dialyze the entire renatured protein in the 50mM KCl buffer. I then run it over a Source Q column in 30mM Tris, 10% glycerol, 1mM DTT, 50mM KCl loading salt. I run a gradient from 150mM to 400mM KCl in 35ml and IIA elutes around 225mM KCl. This preparation is usually free of most contaminants. Some people additionally run the prep over a gel filtration column, which I haven’t needed to do.
RB Buffer
30mM Tris 8.0
2mM EDTA 8.0
10% glycerol
500mM KCl
The expression plasmids are:
pLH41 wt TOA2, 365 bp NcoI/BamHI insert, sequenced in pET21d
pLH44 wt TOA1, 860 bp NdeI/BamHI insert, sequenced in pET21a
