Fractionation of perforin and granzymes by immobilized metal affinity chromatography (IMAC)

Abstract

Cytotoxic lymphocytes and natural killer cells kill their targets by releasing pore-forming granules or by Fas ligand-Fas initiated death. The granules contain the pore-forming protein perforin, proteoglycan and multiple serine proteases termed granzymes. In this paper we describe two options for isolating perforin and granzymes. Both options separate the proteins by their ability to bind to immobilized metal affinity chromatography (IMAC) columns. The first option, with Cu 2+ as the metal (Cu 2+-IMAC), separates both perforin and granzymes while the second, with Co 2+ as the metal (Co 2+-IMAC), separates only perforin. After Cu 2+-IMAC perforin is > 20-fold enriched with excellent recovery of lytic activity. Only two proteins are substantial contaminants. After Cu 2+-IMAC, the perforin is dilute and requires concentration before additional steps of purification. The second option, with Co 2+ as the metal (Co 2+-IMAC), yields perforin that is concentrated in a sharp peak. The concentrated perforin is immediately suitable for further purification. The first option, with Cu 2+, isolates the granzymes while the second option, Co 2+-IMAC, does not. After isolation, the perforin lytic and granzyme activities are stable for weeks at 4°C, an advantage to previous isolation methods for these proteins. The excellent recoveries of perforin and granzymes also indicate that these proteins are less than 4% and 15% of the total lymphocyte granule protein, respectively.