Editing Purification of T4 DNA Ligase (section)

== Three step purification ==

Purification of DNA Ligase from E. coli Strain 594 (Agt4 lop 11 Zig+ S7)-All operations were carried out at 0-4°C. Growth of Cells and Preparation of Extract-E. coli 594 bearing the prophage hgt4 lop 11 Zig+ S7 was grown at 30°C with constant aeration in a New Brunswick fermentor in 80 liters of broth containing 10 g of K2HP04, 1.85 g of KHzPO+ 10 g of Ardamine Z yeast extract, 10 mg of thiamin, 50 mg of thymine, 1% cerelose/liter. The pH was maintained at 7.1 to 7.3 by addition of 50% NaOH. When the density of the culture reached an As95 of 1, the temperature was raised to 42°C for 15 min then lowered to 37°C and aerated for 2 h. The cells (450 g) were harvested in a Sharples centrifuge and resuspended in 50 mM Tris-HCl (pH 7.5) containing 10% sucrose so that a 400-fold dilution of the suspension gave an A595 of 0.5. The resuspended material was quickly frozen and stored at -20°C.

One hundred twenty grams of cell paste were thawed slowly and 180 ml of cold buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol were added. The cells were disrupted by two passages through a Manton Gaulin homogenizer at a pressure of 6500 p.s.i. The extract (300 ml) was clarified by centrifugation at 18,000 x g for 30 min. This fraction (Fraction I) can be stored at -20°C for periods of six months or longer without significant loss of activity.

Polymin P Precipitation and Elution

The amount of polymin P (10% v/v) required to precipitate DNA ligase from the extract was determined by the addition of increasing amounts (0 to 2%, final concentration) to l-ml samples of Fraction I. The samples were mixed thoroughly, kept at 0°C for 5 min, centrifuged at 10,000 X g for 10 min, and the clear supernatant fluid assayed for activity. A final concentration of 0.9% polymin P generally resulted in quantitative precipitation of the enzyme. In the preparation described, 30 ml of a 10% polymin P solution was added to 300 ml of Fraction I. After centrifugation of the suspension, the pellet was resuspended in 150 ml of a solution composed of 20 mM potassium phosphate (pH 6.5), 2 mM EDTA, 1 mM dithiothreitol, and 0.2 M ammonium sulfate, using a Dounce homogenizer. It was then centrifuged at 10,000 X g for 15 min and the supernatant fluid (Fraction II) was treated immediately with ammonium sulfate.

Ammonium Sulfate Fractionation

Solid ammonium sulfate (0.2 g/ml) was added to Fraction II with stirring over a period of 10 to 20 min. After an additional hour of stirring, the suspension was centrifuged at 18,000 x g for 30 min and the precipitate was discarded. Solid ammonium sulfate (0.187 g/ml) was added to the supernatant fluid and the suspension was stirred and centrifuged as before. The pellet was dissolved in 100 ml of 20 mM potassium phosphate (pH 6.5), 2 mM EDTA, 1 mM dithiothreitol, 10 mM ammonium sulfate (Buffer A). The solution (Fraction III) was quickly frozen and stored at -20’. At this stage, the ratio of absorbance at 280 nm to that at 260 nm was 1.15 or greater, indicating that nucleic acid contamination was negligible.

Phosphocellulose Chromatography

Fraction III (75 ml) was dialyzed against 100 volumes of Buffer A containing 15% (v/v) glycerol for 5 h. The precipitate which formed was removed by centrifugation (15,000 X g for 10 min). The supernatant fluid containing 0.9 g of protein was applied to a phosphocellulose column (35 x 6.2 cm) equilibrated with Buffer A containing 15% glycerol. The column was washed with 3 liters of buffer, and then a g-liter linear gradient of KC1 (0 to 100 mM) in the same buffer was applied. The flow rate was 200 ml/h, and 200-ml fractions were collected. DNA ligase eluted at 70 mM KC1 (Fraction IV; 1500 ml) (Fig. 1). Fraction IV was concentrated by absorption to a lo-ml column of DEAE-Sephadex A50 equilibrated with Buffer A containing 70 mM KC1 and 15% glycerol and eluted with the same buffer containing 200 mM KCI. The DNA ligase activity was recovered quantitatively in a single broad peak (60 ml). It could be further concentrated (up to 10 mg/ml or more) by precipitation with ammonium sulfate at 70% saturation.
[[Category:Protocols]] [[Category:Protein Purification]]