Cell-targeted lytic pore-forming agents

DETAILED DESCRIPTION The drawings are first briefly described.
DRAWINGS FIGS.
1a-1c are a set of graphs showing activation of hemolytic activity in overlap mutants of .
alpha.
HL by protease treatment.
A key to structures of two-chain .
alpha.
HL complementation mutants produced by coupled in vitro transcription/translation (IVTT) is shown in the left panel.
The center and right panels show the results of a microtiter assay using two-fold serial dilutions.
Assays of mutants which have not been treated with protease are shown in the center graph and assays of replicate samples after treatment with endoproteinase Lys-C (endo C) are shown in the right graph.
In each window, the tops of the bars indicate the well with 50% hemolysis after 1 h, 3 h and 24 h (left to right).
The top of each window represents well 12 and the bottom well "0" ("0" denotes no hemolysis in well 1).
The vertical scale is logarithmic to the base 2.
Overlap mutants were activated by adding endo C (1.
0 .
mu.
g) to the IVTT mix (10 .
mu.
L).
The initial dilution of the IVTT mix in well 1 was 1:4.
The individual polypeptides that make up the two-chain mutants have no hemolytic activity.
FIG.
2 is a set of graphs showing hemolysis by selected mutants as determined by a spectrophotometric assay.
The decrease in light scattered at 600 nm, an indication of erythrocyte lysis, was monitored after the addition of IVTT mix (10 .
mu.
L, before additions) to 0.
025% rabbit erythrocytes (500 .
mu.
L).
The mutants used are as in FIGS.
1a-1c, with the addition of the non-activatable gap mutant .
alpha.
HL(1-131).
circle-solid.
(143-293) as a control (bottom left panel).
FIGS.
3a-3b are photographs of endo-C-treated proteolytic fragments of overlap mutants and their component polypeptides separated using sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE).
FIG.
3a shows untreated individual chains and FIG.
3b shows individual chains treated with endo C (1 .
mu.
g added to 6 .
mu.
L IVTT mix).
FIGS.
4a-4b are photographs of an SDS-PAGE gel showing the results of treatment of the two-chain mutants with endo C