Calcium hydroxyapatite fractionation as a second ion exchange step in protein purification: Improvement of flow rate and resolution

The use of washed brushite, CaHPO 4, as a filter aid permitted the flow rates of calcium hydroxyapatite (HAP) columns to be made reliable and high. Regeneration of the column contents yielded pure, crystalline HAP, which could be mixed with fresh brushite and reused. Column fractionation over HAP-brushite with a phosphate gradient was used as a second ion exchange step in protein purification, the first step being fractionation over DEAE-cellulose. In two examples discussed, HAP-brushite fractionation in the presence of an effector molecule, a molecule for which the enzyme of interest had a specific affinity, gave marked improvement of resolution.