Purification of a-actinin from Acanthamoeba

Dyche Mullins 11/98

1. Follow general protocol for amoeba prep using sucrose buffers. Elute protein from DEAE column with 0.0-0.6 M linear gradient of KCl in ATP- and calcium-containing buffer.

2. Collect and pool gel-ed fractions containing actin and a-actinin. These usually have conductivity between 7-11 mMHO or KCl concentration around 0.3 M. Conscientious method: Determine positions of actin and a-actinin by Elisa. Dirtball method: Allow fractions to sit for 1-2 days in the cold room and pool obviously gel-ed fractions

3. Add 2 mM MgCl₂ and warm fractions to room temperature to polymerize actin. Incubate 1 hour at 24 C with stirring.

4. Spin down actin for 90 minutes at 4 C at 38,000 RPM in Ti-45 rotor. Keep pellets for actin prep. Take sups for a-actinin prep.

5. Add solid ammonium sulfate to 1 M and incubate with stirring at 4 C for 4-6 hours. Spin out precipitate at 3,000 RPM for 30 minutes. Retain supernatant. Keep pellet just in case.

6. Add additional ammonium sulfate to supernatant to a total concentration of 2.3 M and incubate with stirring overnight. Spin out precipitate at 3,000 RPM for 30 minutes. Keep supernatant just in case.

7. Resuspend pellet in 0.8 M KCl, 10 mM Imidazole (pH 7.5), 1 mM CaCl₂, 1 mM DTT, 0.01% NaN₃. Dialyze vs. this buffer o/n with 1-2 changes.

8. Add solid KI to 0.6 M and homogenize the sample in a Dounce homogenizer with a tight-fitting plunger. Spin at 38,000 RPM in Ti-45 rotor for 30 minutes at 4 C.

9. Run on 4x100 cm Sephacryl S-300 equilibrated with 0.6 M KI, 10 mM Imidazole (pH 7.5), 1 mM DTT, 0.01% NaN₃. Assay fractions for a-actinin by SDS-PAGE, ELISA, or falling-ball viscometry and pool. Alternately, on a calibrated column take the fractions corresponding to a Stoke's radius of 8.5 nm.

10. Dialyze 3-4X vs. 1L of 10 mM Imidazole, 1 mM DTT, 0.01% NaN₃. Load onto 2.5 x 7 cm column of hydroxlapatite (BioRad HT) equilibrated with the same buffer. Elute with a linear gradient of 0-100 mM K-phosphate in a total of 300 mL column buffer. Assay fractions by SDS-PAGE or falling ball viscometry. a-actinin usually comes out in the first peak. Dialyze vs 1 L 10 mM Imidazole (pH 7.5), 1 mM DTT, 0.01% NaN₃. After this step the a-actinin is clean enough for most purposes.

11. To remove the final minor contaminants, run the a-actinin on a 2.5 x 7 cm column of DEAE and elute with a linear gradient of 0-400 mM KCl in a total volume of 300 ml column buffer.