Isolation of a lytic, pore-forming protein (perforin) from cytolytic T-lymphocytes

Abstract

Cytolytic granules from a T-cell line with specific cytolytic activity were isolated. Granules were solubilized and fractionated on a TSK 4000 gel filtration column. Lytic activity was eluted as a single retarded peak. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the lytic fractions contained a single protein (perforin) with an apparent molecular weight of approximately 66 kDa. It separated well from the other proteins present in the granules. Isolated perforin polymerized and inserted into lipid bilayers in the presence of Ca2+, forming tubular structures with inner diameters varying from 6 to 16 nm. Lipid insertion of perforin was demonstrated using a membrane-restricted, photoactivatable probe. The lytic properties of perforin suggest an important role of this particular protein during cytolytic T-cell-mediated target cell lysis.