EXTRACTION AND CHARACTERIZATION OF THE INSECTICIDAL TOXIN HIRSUTELLIN-A PRODUCED BY HIRSUTELLA-THOMPSONII VAR THOMPSONII

Hirsutellin A (HtA) produced by Hirsutella thompsonii var. thompsonii (strain JAB-04) was extracted and purified using a combination of ion- exchange, gel-permeation, and immunoaffinity chromatography. The ident ity of the purified HtA was confirmed by amino acid analysis and N-ter minal sequencing. Monoclonal antibodies prepared against HtA were capa ble of detecting 25-50 ng of HtA by direct sandwich ELISA. In addition , utilizing Western blot methods, the antibodies were shown to be spec ific to HtA. The production of HtA was monitored during submerged ferm entation. The peak level of exocellular HtA (13-14 mu g/ml) was during the late exponential growth phase (39-45 h), determined by utilizing a combination of densitometric analysis of the 16.3-kDa bands on SDS-P AGE gels and ELISA. HtA production was directly correlated with myceli al growth. Twenty-one-hour culture filtrates were highly toxic re, lar vae of the greater wax moth. Pure HtA at a final concentration of 40 p mol was highly toxic to Galleria mellonella larvae. (C) 1995 Academic Press, Inc.