HUMAN PERFORIN - RAPID ENRICHMENT BY IMMOBILIZED METAL AFFINITY-CHROMATOGRAPHY (IMAC) FOR WHOLE-CELL CYTOTOXICITY ASSAYS

Perforin, a potent pore-forming protein, plays an important role in ly mphocyte-mediated cytolysis causing necrosis or, when combined with th e granzymes, apoptosis. The studies on perforin, although already exte nsive, have been hampered by the limited amount of material available from killer lymphocytes. Using a cell Line that expresses high levels of human perforin, we describe a straightforward purification scheme t hat allows isolation of the lytic protein in approximately 8 hours. Pe rforin is enriched from the YT-INDY cell line by cavitation followed b y differential centrifugation and ion metal affinity chromatography. I n addition to demonstrating the lytic activity of human perforin towar d various cell lines, we show that the conditions of the cytotoxicity assay influence both the kinetics and magnitude of perforin-mediated c ytotoxicity. (C) Academic Press, Inc.