The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth.

A 67-kDa protein that can specifically bind the activated Cry9A endotoxin under ligand-blotting conditions was purified from midgut epithelium apical membranes of wax moth Galleria mellonella by affinity chromatography. N-Terminal amino acid sequencing enabled identification of this protein as aminopeptidase N. In similar experiments, 66- and 58-kDa proteins specific to endotoxin Cry3A were isolated from the midgut epithelium apical membranes of Tenebrio molitor larvae. Mass spectrometry showed close similarity of the 58-kDa protein to the Tenebrio molitor α-amylase.

An expression system for an effective production of recombinant protein Cry9A in bacillary cell has been suggested in the study. The proteins’ immunological properties, ability to proteolysis, and biological activity were identical to natural protein. The ability of recombinant Cry9A to form crystal bodies in sporulating cells of Bacillus thuringiensis has been shown. Thus, the first evidences of the fact that Cry-proteins which in natural strains form the crystal bodies together with other endotoxins are able to independently form the crystals has been received. The introduced system including vector replicative carriers, expression cassettes, and a protocol of obtaining and cultivation of strain-producer allows simple manipulations with the gene of delta-endotoxin of Cry9A in gene-engineering experiments.