Cytolytic activity of purified recombinant perforin. (A) Human wild-type and A91V mutant perforins were purified using a baculovirus system; their respective concentrations were equilibrated using Western blots and the cytolytic activity tested using SRBC lysis assay or 51Cr release from EL-4 cells (mean ± SD). (B) Purified recombinant hPRF-A91V and hPRF-WT proteins were mixed at 0:1, 0.2:1, 0.4:1, 0.8:1, 1.6:1, 2.4:1, 4:1, and 12:1 ratios and then added to SRBCs. The large graph shows nonstandardised (raw) levels of SRBC lysis in which 69% ± 4% lysis corresponds to the addition of hPRF-WT alone to the cells. The data are shown as mean ± SE of 3 to 4 independent experiments and performed with 2 independent batches of perforin. The small graph depicts standardized values shown on the large graph, where “control” was designated as 100%. (C) Purified recombinant WT and A91V perforins were subjected to the gel filtration, and the elution profile was analyzed using Western blots (see “Western blotting” in “Materials and methods” for details).
