| 50 ml of Buffer A (10 mM Tris base, 0.1 M Na2HPO4, 0.1% Nonidet P-40, 10 mM B-mercaptoethanol, 100 mM NaC1, 10% glycerol, 4 M urea)/pH 7.8, |
| The cells were solubilized in 50 ml of Buffer A (10 mM Tris base, 0.1 M Na2HPO4, 0.1% Nonidet P-40, 10 mM B-mercaptoethanol, 100 mM NaC1, 10% glycerol, 4 M urea)/pH 7.8, with slow shaking for 1 h at room temperature. The slurry was centrifuged at 12,000 X g for 10 min, and the supernatant slowly applied to a 1-ml nickel nitrilotriacetate affinity column (Ni-NTA, Qiagen, Chatsworth, CA) pre-equilibrated with Buffer A/pH 7.8, at 4c.
All manipulations from this point were performed at 4c. The column was washed with 10 column volumes of Buffer A/pH 7.8, followed by 10 column
volumes of Buffer A/pH 6.3, to remove the majority of background E. coli proteins prior to elution of IN. These steps were followed by 10 column volumes of Buffer A/pH 5.9, and 10 column volumes of Buffer A/pH 4.5. IN protein eluted after 1 column volume of Buffer A/pH 4.5. Protein concentration was measured by the method of Bradford (24) (Bio-Rad).
An alternative protocol, purification B, was developed which substituted the B-mercaptoethanol in Buffer A with dithiothreitol, designated Buffer B. A 10-ml Ni-NTA column was developed as above with stepwise pH elutions after the application of crude extracts of pETllC or pETINH1. The majority of the E. coli proteins eluted in the first wash with Buffer B/pH 7.8. IN protein eluted throughout the first 6 column volumes of the Buffer B/pH 6.3 elution step, and the first four fractions of the Buffer B/pH 5.9 elution step. No IN protein was eluted in the Buffer B/pH 4.5 step. In all purifications, column fractions were slowly renatured by a 24-h step dialysis from 3 to 0 M urea in 2 liters of 20 mM HEPES, pH 7.4,1 mM dithiothreitol (DTT), 20% glycerol, 0.1% Nonidet P-40, 400 mM monopotassium glutamate over 4 days. Fractions were frozen in liquid nitrogen and stored at -70 C. A specific activity of 2.2 units/mg was calculated for the pH 5.9 fraction purified in prification B. One unit is defined as 1 pmol of U5 substrate converted to autointegrants/min. |