Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraeamylitta

Abstract

An inhibitor of Aspergillus oryzae fungal protease was purified to homogeneity from the hemolymph of fifth instar larvae of Antheraea mylitta by ammonium sulfate precipitation, anion exchange and gel filtration (FPLC) chromatography, and termed as AmFPI-1. The extent of purification was checked by two-dimensional gel electrophoresis, and the molecular weight of purified inhibitor was determined by SDS-PAGE as 10.4 kDa. Fifteen N-terminal amino acid sequences of this protein were determined, and degenerate oligonucleotides were synthesized on the basis of these sequences. A cDNA library of A. mylitta integument was constructed, and protease inhibitor cDNA was partially amplified by PCR using degenerate oligonucleotides and CDS primers. A full-length inhibitor cDNA clone obtained by screening the library with PCR amplified DNA as probe was sequenced. The cDNA consists of 543 nucleotides with an ORF of 315 bp and encodes a protein of 105 amino acids. The sequence exhibits similarity to several Bombyx mori ESTs, and in particular to N-terminal amino acid sequence of an inducible serine protease inhibitor (ISPI-1) from Galleria mellonella indicating its relatedness to ISPI-1 of G. mellonella. The presence of this protease inhibitor in the hemolymph may play an important role as a natural defense system against invading microorganisms.