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Revision as of 15:07, 26 January 2009
E. coli ClpP protein with a C-terminal His6 tag was expressed in E. coli strain DH5α/pYK133/pRep4.
Cultures were grown at 30°C to an OD600 of 0.5 in LB with antibiotics, and IPTG was added to 0.5 mM.
Cells were harvested by centrifugation 2–3 hr later and resuspended in 3 ml of S buffer per gram of cells.
Following sonication, the lysate was centrifuged for 20 min at 17,000 × g, and the supernatant (about 30 ml) was added to 1.5 ml nickel-NTA resin (Qiagen), preequilibrated with S buffer.
After mixing for 1 hr at 4°C, the resin was packed into a column and washed with 200 ml S buffer and 100 ml W20 buffer.
Protein was eluted with W500 buffer, and fractions containing ClpP were pooled and
desalted into Q50 buffer.
This material was loaded onto a MonoQ HR5/5 FPLC column (Pharmacia) and eluted with a 20 ml gradient from Q50 to Q1000 buffer.
Fractions containing ClpP were pooled, dialyzed against Clp buffer at 4°C, and stored at −80°C. Concentrations of ClpP14 and ClpX6 were determined by UV absorbance (e = 125160 M-1 cm-1)
- PD buffer: contains 25 mM HEPES-KOH (pH 7.6), 5 mM KCl, 5 mM MgCl2, 0.032% NP-40, 10% glycerol, 5 mM ATP, and an ATP-regenerating system consisting of 16 mM creatine phosphate and 0.32 mg/ml creatine kinase.
- Clp buffer: 50 mM Tris-HCl (pH 7.5), 200 mM KCl, 25 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, and 10% glycerol.
- S buffer: 50 mM Na-phosphate (pH 8.0), 1 M NaCl, 5 mM imidazole, and 10% glycerol.
- W20 buffer: 50 mM Na-phosphate (pH 8.0), 1 M NaCl, 10% glycerol, and 20 mM imidazole;
- W500 buffer is the same but contains 500 mM imidazole.
Q50 buffer: 50 mM Tris (pH 8.0), 5 mM DTT, 10 mM MgCl2, 10% glycerol, and 50 mM KCl;
Q1000 buffer is the same but contains 1 M KCl.