PURPOSE: To efficiently purify a protein such as an enzyme by treating the protein with the chromatography of a hydroxyapatite in two stages comprising a stage having the ligand of the protein and the other not having the ligand.

CONSTITUTION: When a protein is purified with chromatography using a hydroxyapatite, the treatment of the chromatography is performed in two stages to effectively purify the protein. The two stages comprises a stage subjected to a conventional method and the other stage subjected to a condition using the substrate of the protein to be purified, an activating agent, allosteric effector, their analogous compounds, and further a specific ligand such as an inhibitor. The same effect is obtained even when either of the two stages is first performed or even when another purification means is nipped with the two stages. When an enzyme for synthesizing δ-aminolevulinic acid is purified (using ATP as a ligand) by the method of this invention, the rate of the purification can be improved larger 21 times than by a conventional method, and the scale of the subsequent purification stage can be reduced into ≤l/25.