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Site-Directed Mutagenesis and Purification of His-Tagged ResT. Wild-type and mutant ResT genes were cloned into the plasmid vector pET15b (Novagen) and flanked by a 6´His-tag at the N terminus. Mutant resT genes were generated by using the QuikChange site-directed mutagenesis protocol (Stratagene), except that Deep Vent polymerase (NEB, Beverly, MA) was used. Transformed Escherichia coli Rosetta BL21 (DE3 pLysS pRARE) cells (250 ml) carrying the gene encoding for either wild-type or mutant ResT were grown in Luria broth supplemented with ampicillin (100 mg/ml; EM Science) and chloramphenicol (30 mg/ml; EM Science) at 37°C to midlog phase (A₅₉₅ = 0.5). ResT expression was induced with a final concentration of 0.75 mM isopropyl b -D-thiogalactopyranoside (EM Science), after which cultures were shaken at 18°C overnight. Cells were pelleted by centrifugation at 5,400 ´g for 6 min at 4°C, and cell pellets were resuspended in 4 ml of resuspension buffer (25 mM Hepes-NaOH, pH 7.6/0.2 mM EDTA, pH 8.0). Solid KCl was added to the resuspended cells to a final concentration of 0.8 M, followed by the addition of 375 ml of protease inhibitor mixture (Sigma). The resuspended cells were then subjected to a single freeze–thaw cycle, after which 450 ml of lysozyme (25 mg/ml; EM Science) was added and allowed to sit on ice for 30 min. The lysozyme-treated cells were then put through three additional freeze–thaw cycles before ultracentrifugation at 90,000 ´g (Beckman 55.2TI rotor) for 1 h at 4°C.

Supernatants were decanted into 50-ml tubes, and glycerol was added to 10% (wt/vol). Columns for His-tag affinity purification were prepared by pipetting 1.5 ml of Ni-nitrilotriacetic acid slurry (Qiagen, Valencia, CA) and equilibrating with 8-10 ml of loading buffer [50 mM NaH₂PO₄/300 mM NaCl/10 mM imidazole/10% glycerol wt/vol (adjusted pH to 8.0)]. Loading buffer without NaCl was added to the lysate to achieve a final salt concentration of 300 mM. The diluted lysates were then loaded onto the Ni-nitrilotriacetic acid column and washed with 10-15 ml of wash buffer [50 mM NaH₂PO₄/ 300 mM NaCl/20 mM imidazole/10% glycerol wt/vol (pH adjusted to 8.0)]. His-tagged ResT was eluted with elution buffer [50 mM NaH₂PO₄/300 mM NaCl/250 mM imidazole/10% glycerol wt/vol (adjusted pH to 8.0)], and fractions were collected. Samples were subjected to SDS/PAGE, and fractions containing purified His-tagged ResT were identified through staining with Coomassie blue.