Purification of Mitochondrial Uracil-DNA Glycosylase Using Ugi-Sepharose Affinity Chromatography

Affinity chromatography is a powerful technique that allows purification of proteins based on biospecific interactions using an affinity absorbent to selectively bind a target protein (1 ,2 ). Separation is achieved through a highly specific, but reversible, interaction with a complementary binding substrate (ligand) that is immobilized on a solid support (matrix). This purification approach has been developed to incorporate protein-protein interactions into the design of the affinity matrix (3 –3 ). Affinity chromatography procedures can achieve purification levels on the order of several thousand-fold in a single separation step. The method presented here utilizes the bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein as an immobilized ligand on Sepharose 4B beads for purification of mammalian mitochondrial uracil-DNA glycosylase.