CHARACTERIZATION OF RECOMBINANT HUMAN ALDOLASE-B AND PURIFICATION BY METAL CHELATE CHROMATOGRAPHY

Recombinant human aldolase B and the native enzyme purified from human liver were found to be identical in size, charge, structure, K-m cons tants for fructose-1,6-bis(phosphate) and fructose-1-phosphate, and th e activity ratio of the two substrates. Thus recombinant aldolase B is a valid model for the native enzyme and can be used to study mutation s that cause hereditary fructose intolerance or others designed in the active site. Addition of six histidine residues to the amino-terminus of the recombinant enzyme did not alter its structural or functional characteristics and allowed for purification by immobilized metal affi nity chromatography. This purification protocol does not require a sta ble or active enzyme and will facilitate the study of mutant aldolase B enzymes that would otherwise be difficult to purify. (C) Academic Pr ess, Inc.