Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella

Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purifie d from larval hemolymph of greater wax moth larvae, Galleria mellonella, an d characterized at a molecular level. These inhibitors were synthesized aft er larvae were injected with a yeast polysaccharide, zymosan preparation. I SPI-1,2,3 were active against various serine proteases including trypsin an d toxic proteases released by the entomopathogenic fungus Metarhizium aniso pliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitor s from cell-free hemolymph samples. The molecular masses of purified protei ns were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 k Da (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-term inal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other kno wn proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.