HA22 mutants. A series of deletions within domains II and Ib were introduced into the PE38 component of HA22 to eliminate lysosomal protease cleavage sites. These 5 mutant proteins (M1, M2, M3, M4, and M5) are illustrated using an expanded view of domains II and Ib of PE38 to show the extent of the deletions (dotted lines) and the presence of the C287S point mutation. Residue numbering is based on the location of amino acids in native PE. The proteins were subsequently purified and compared with HA22 using an in vitro cytotoxicity assay on Raji cells. The M5 protein was renamed HA22-LR for further analysis. The IC₅₀ (ng/mL) of each mutant relative to the IC₅₀ of HA22 is presented as a mean of at least 3 separate experiments.