Editing Purification of T4 DNA Ligase (section)

== Protocol 1 ==

Preparation of Cell Extract

Unless otherwise noted, all steps were performed at 4°C. One hundred grams of frozen cells were thawed and resuspended in 400 ml of 10 mM K2HPO4/KH2PO, pH 7.5, 1 mM EDTA, 1 mM glutathione (PEG buffer) containing 0.4 M NaC1, 25 pg/ml of phenylmethylsulfonyl fluoride, and 100 pg/ml of lysozyme. After 10 min at O"C, the cell suspension was sonicated with a Bronwill Biosonik I11 sonifier set at maximum output for the 1-inch probe. Sonication was in four 30-s pulses at 3-min intervals with the suspension stirring in a salt-ice bath; the temperature remained below 8°C at all times. The sonicated suspension was centrifuged for 1 h at 35,000 rpm, at 4°C in the Beckman type 35 rotor. The supernatant was decanted, measured, diluted with 1.25 volumes of cold PEG buffer, and applied directly to a column of phosphocellulose.

Phosphocellulose Chromatography

A phosphocellulose column (4 X 35 cm) prepared as described in Greene et al. (8) was equilibrated in PEG buffer containing 0.2 M NaCI. The diluted extract was loaded directly onto the column and then washed with the same buffer until the A~w was near 0. The column was developed with a 2-liter gradient of 0.2 to 1.0 M NaCl in PEG buffer. Fifteen-milliliter fractions were collected and assayed for ligase activity. As shown in Fig. 1, ligase activity eluted at 0.5 to 0.6 M NaCI. Peak fractions (55 to 72) were pooled, measured, and diluted with an equal volume of 10 mM KzHP04/KH2PO4 pH 7.5, 10 mM P-mercaptoethanol, 5% glycerol (PBG buffer).

Hydroxylapatite Chromatography-A 30-ml bed volume column of hydroxylapatite was poured and equilibrated with PBG buffer containing 0.05 M KCI. The diluted phosphocellulose pool was applied to the column and the column was washed with this buffer until the A,, was near 0. DNA ligase was eluted with a 200-1111 gradient of 0 to 10% ammonium sulfate in the same buffer. Three-milliliter fractions were collected and assayed for ligase activity, shown in Fig. 2. Peak fractions (27 to 45) were pooled and dialyzed against PBG + 0.05 M KC1 for 2 h to dilute the ammonium sulfate concentration. Dialysis was continued for 12 h against 10 mM K2HPO4/KH2PO, pH 7.5.0.05 M KCI, 10 mM P-mercaptoethanol, 0.5 mM NaN.r, 50% glycerol, and the concentrated ligase was stored at -20°C.

Cohesive-end and Blunt-end Ligation of DNA Fragments

DNA ligase activity was also assayed for its ability to ligate cohesive and blunt-end DNA restriction fragments. For the cohesive-end ligation assay, 50-4 reaction mixtures containing 20 mM Tris-HCI, 10 mM MgC12, 10 mM dithiothreitol, 0.6 mM ATP, 1 pg of HindIII-digested pBR322 DNA, and 1 pl of a serial dilution of purified DNA ligase were incubated at 14°C for 1 h. Each reaction was terminated by the addition of 30 p1 of 0.05% xylene cyanol, 0.05% bromphenol blue, 10 M urea (XC dye). and 16 pI of the mixture applied to a 1% agarose gel. Gel electrophoresis was performed as previously described (9). Blunt-end ligation was assayed in lop1 of the same reaction mixture with the exception that Hue 111-digested pBR322 DNA was used instead of HindIII-cleaved DNA. In this case, the reactions were incubated at 23°C for 1 h. The reactions were terminated by the addition of 15 pI of XC dye and 12.5 pl of this mixture was taken for analysis on a 7.5% polyacrylamide gel.

Endonuclease Assay

The presence of deoxyendonucleases in the purified ligase was determined by incubating 0.2 pg of undigested pBR322 DNA with 1 p1 of DNA ligase (4 pg of protein) at 37°C from 1 to 5 h. Each 30-pl reaction mixture contains the same buffer described above. The reactions were terminated with 10 pl of XC dye and the total volume was analyzed by 1% agarose gel electrophoresis The specific activity of the ligase was determined to be 70 units/pl of ligase, approximately 17,500 units/mg of protein, with a total yield of 800,000 units of ligase activity. In contrast, it has been reported that 45 g of T4-infected cells yielded 1200 units of homogeneous DNA ligase activity with a specific activity of 2,500 units/mg