Paul.xu: Created page with "== 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 m..."

2017-06-16T17:15:09Z

Created page with "== 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 m..."

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== 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

== 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]]

Purification of T4 DNA Ligase - Revision history

Paul.xu: Created page with "== 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 m..."