FRACTIONATION OF PERFORIN AND GRANZYMES BY IMMOBILIZED METAL AFFINITY-CHROMATOGRAPHY (IMAC)

Cytotoxic lymphocytes and natural killer cells kill their targets by r eleasing pore-forming granules or by Fas ligand-Fas initiated death. T he granules contain the pore-forming protein perforin, proteoglycan an d multiple serine proteases termed granzymes. In this paper we describ e two options for isolating perforin and granzymes. Both options separ ate the proteins by their ability to bind to immobilized metal affinit y chromatography (IMAC) columns. The first option, with CU2+ as the me tal (Cu2+-IMAC), separates both perforin and granzymes while the secon d, with Co2+ as the metal (Co2+-IMAC), separates only perforin. After Cu2+-IMAC perforin is > 20-fold enriched with excellent recovery of ly tic activity. Only two proteins are substantial contaminants. After Cu 2+-IMAC, the perforin is dilute and requires concentration before addi tional steps of purification. The second option, with Co2+ as the meta l (Co2+-IMAC), yields perforin that is concentrated in a sharp peak. T he concentrated perforin is immediately suitable for further purificat ion. The first option, with CU2+, isolates the granzymes while the sec ond option, Co2+-IMAC, does not. After isolation, the perforin lytic a nd granzyme activities are stable for weeks at 4 degrees C, an advanta ge to previous isolation methods for these proteins. The excellent rec overies of perforin and granzymes also indicate that these proteins ar e less than 4% and 15% of the total lymphocyte granule protein, respec tively.