I. 3-AB affinity resin synthesis

ECH Sepharose 4B is formed by covalent linkage of 6-aminohexanoic acid to Sepharose 4B using an epoxy coupling method. ECH Sepharose has free carboxyl groups at the end of a 6-carbon spacer arms, which are used to couple ligands containing primary amino groups with the carbodiimide coupling method. The long flexible hydrophilic spacer arm connected to the gel is particularly suitable for immobilization of small molecules such as inhibitor containing free NH₂ groups like 3-aminobenzamide (3-AB).

Materials :

ECH Sepharose 4B (Pharmacia Biotech) 50 ml (#17-0571-01)
N’-(3-dimethylaminopropyl)-N-ethylcarbodiimide Hydrochloride, 98% (Sigma-Aldrich) 10 g (#16,146-2)
3-Aminobenzamide (3-AB), 99%, (Sigma-Aldrich) 1 g (#A-0788)

Protocol description :

The method for coupling is the carbodiimide method. The N,N’-disubstitued carbodiimide promotes condensation between the free amino of 3-AB and a free carboxyl group to form a peptide link by acid catalyzed removal of water.

1. Preparing the gel: ECH Sepharose 4B is supplied preswollen in 20% ethanol. Decant the ethanol and wash the required amount of gel (25 ml) on a sintered glass filter with about 80 ml 0.5 M NaCl per ml sedimented gel added in several aliquots.

2. Coupling the 3-AB: The coupling reaction is performed in the cold room at 4 °C during 12 to 24 hours in distilled water adjusted to pH 4.5 - 6.0 with 0.1 M sodium hydroxide to promote the acid-catalyzed condensation reaction. The pH value of the reaction mixture decreases during the first hour of the coupling. The pH must therefore be adjusted during this time by addition of 1 N sodium hydroxide solution. The reaction is performed in a 50 ml Falcon tube containing 25 ml of the gel. The volume of the solution is adjusted to 50 ml with water. 1 g of N’-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride is added plus 0.5 g of 3-AB dissolved in 1 ml of methanol 100%. Rotate, using a rotating wheel, the mixture end-over-end overnight in the cold room. During the &Mac222;rst hour, control and adjust the pH if necessary.

3. Stop the coupling reaction: The excess of ligand is eliminated by washing the gel thoroughly in the buffer containing 100 mM acetate pH 4.0 and 0.5 M NaCl, then in the buffer containing 100 mM Tris-HCl pH 8.0 and 0.5 M NaCl. Wash with distilled water. The column can now be packed and equilibrated. The column is stable for many years and can be used many times if kept in the appropriate conditions (at 4 °C in buffer containing 0.02% sodium azide).

Comments : This method of purification using the ECH Sepharose 4B has been found much more efficient than the previous method using the Affigel-10 from Biorad which for unknown reason had a lower stability in time. We found this column coupled to 3-AB very useful for affinity purification of PARP-1, -2 and –3. It should be potentially usable for the purification of other members of the PARP family displaying a good affinity for 3-AB.

II. PARP-1 purification protocol from insect cells

The following protocol is suitable for purification of the human recombinant PARP-1, -2 and -3 overexpressed in Sf9 insect cells (Amé et al., 1999 ; Augustin et al., 2003 ; Giner et al., 1992). This system results in a high level protein expression and only one purification step is usually necessary to obtain high purity protein that can be used for activity assays or even crystallographic studies (Oliver et al., 2004). This protocol can be easily adapted to other expression systems. The purified PARP-1 displays the same catalytic property compared to the classical purification scheme (Zahradka and Ebisuzaki, 1984). It is free of DNA, of topoisomerase I activity and of inhibitor contamination.

Materials :

Proteases inhibitor cocktail tablets (Complete Mini, Roche Diagnostic Gmbh, Mannheim, Germany)
3-methoxybenzamide (Sigma-Aldrich), 5 g, (#M1.005-0)

Protocol description :

Cell lysate preparation

All the steps are performed at 4 °C ideally in the cold room or in ice.

1. Following the expression of the protein, the cells (3 x 10⁹ cells) are harvested and frozen at -80 °C. The cells are then quickly defrosted and resuspended in 5 ml per 10⁸ cells of 25 mM Tris-HCl pH 8.0 buffer containing 50 mM glucose, 10 mM EDTA, 1 mM phenylmethansulfonylfuorid (PMSF), 2 proteases inhibitor cocktail tablets.

2. Add 0.2% of Tween 20, 0.2% of NP40, 1 M NaCl (weigh the necessary amount) final. For 1.5 x 10⁹ cells, the final volume should be 75 ml.

3. Gently agitate the cell suspension for 20 min on a rotating wheel in the cold room. Transfer to a cold glass container.

4. Moderately sonicate 4 x 20 sec on ice. Don’t stop agitating during the sonication process to ensure a good lysis. The viscosity of the solution should disappear.

5. Clear lysate from cellular debris by centrifugating at 50,000 x g for 45 min at 4 °C in polycarbonate tubes (rotor Ti70 Beckman).

6. Add to the supernatant 1 mg/ml final of a 10 mg/ml protamine sulfate solution in water to precipitate the nucleic acids. Centrifuge at 50,000 x g for 20 min at 4 °C in polycarbonate tubes (rotor Ti70 Beckman).

7. Keep the supernatant and slowly add 0.226 g/ml (40%) of ammonium sulfate at 0 °C, mix gently for 30 min. Centrifuge at 20,000 x g for 20 min at 4 °C in polycarbonate tubes (rotor Ti70 Beckman) to eliminate the precipitated proteins.

8. To the supernatant add 0.187 g/ml (70%) of ammonium sulfate at 0 °C, mix gently for 30 min. At this step PARP-1 (and PARP-2) will precipitate. Centrifuge at 20,000 x g for 20 min at 4 °C in polycarbonate tubes (rotor Ti70 Beckman) and discard the supernatant. The pellet is kept on ice until re-solubilization.

ECH Sepharose 4B-3AB affinity purification of PARP-1

1. Equilibrate the column with 4 bed volumes (4 x 50 ml) of the BB-buffer made of 100 mM Tris-HCl pH 7.5, 14 mM beta-Mercaptoethanol, 0.5 mM EDTA and 0.5 mM PMSF.

2. The 70% ammonium sulfate pellet of proteins (from 3 x 10⁹ cells) containing PARP-1 is resuspended in 100 ml per 10⁹ cells of BB-buffer supplemented with 100 mM NaCl.

3. The column is loaded overnight at a low flow rate (10 ml/h) using a peristaltic pump. The flowthrough is collected for further analysis.

4. The column is then washed with 4 bed volumes with successively 100 mM, 400 mM and 800 mM NaCl-containing BB-buffer. The &Mac223;ow rate is adjusted to 50 ml/h. Start collecting individual fractions.

5. PARP-1 is eluted with 4 bed volumes of 400 mM NaCl BB-buffer containing 1 mM 3-methoxybenzamide freshly prepared from a 600 mM stock solution in 100% methanol.

The column is regenerated by washing with 3 bed volumes of BB-buffer.

References :

Amé, J. C., Rolli, V., Schreiber, V., Niedergang, C., Apiou, F., Decker, P., Muller, S., Hoger, T., Ménissier-de Murcia, J., de Murcia, G. (1999). PARP-2, a novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J. Biol. Chem. 274, 17860-17868.

Augustin, A., Spenlehauer, C., Dumond, H., Ménissier-de Murcia, J., Piel, M., Schmit, A. C., Apiou, F., Vonesch, J. L., Kock, M., Bornens, M., de Murcia, G. (2003). PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression. J. Cell Sci. 116, 1551-1562.

Giner, H., Simonin, F., de Murcia, G., Ménissier-de Murcia, J. (1992). Overproduction and large-scale purification of the human poly(ADP- ribose) polymerase using a baculovirus expression system. Gene 114, 279-283.

Oliver, A.W., Amé, J.C., Roe, S.M., Good, V. de Murcia, G., Pearl., L.H. (2004). Crystal structure of the catalytic fragment of murine poly(ADP-ribose) polymerase-2. Nucleic Acids Res. 32, 456-464.

Zahradka, P., Ebisuzaki, K. (1984). Poly(ADP-ribose) polymerase is a zinc metalloenzyme. Eur. J. Biochem. 142, 503-509.