Binding of Bacillus thuringiensis delta-endotoxins Cry1Ac and Cry1Ba to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells

A 120-kDa protein was purified from brush border membrane vesicles of the t ortricid moth Epiphyas postvittana (Walker) based both on its activity as a n aminopeptidase and the ability to bind the Bacillus thuringiensis delta-e ndotoxin Cry1Ac. The purified enzyme had a pi of 5.6 and was a leucine amin opeptidase, with some isoleucine, phenylalanine and tryptophan aminopeptida se activity. Further characterisation showed that the protein was also able to bind Cry1Ba. During purification, the molecular weight of the protein d ecreased from 120 to 115 kDa due to the loss of a glycophosphatidinyl ancho r. The protein was N-terninally sequenced and, using this information and c onserved regions within other insect aminopeptidase-N (APN) sequences, redu ndant primers were designed to amplify the aminopeptidase coding sequence f rom E. postvittana midgut cDNA. The predicted protein sequence from the ful l-length cDNA was most closely related to the APN protein sequence from Hel iothis virescens (61% identity) and shared other features of insect APNs in cluding a Zn2+ binding site motif and four conserved cysteines. The E. post vittana was expressed in Sf9 cells using baculovirus, yielding a protein of molecular weight 130 kDa, but with unchanged N-terminal sequence. Purified recombinant protein bound both Cry1Ac and Cry1Ba by ligand blot assays. Ho wever, despite the protein being expressed on the external surface of the S f9 cells, it bound neither Cry1Ac nor Cry1Ba in vivo. (C) 2000 Elsevier Sci ence Ltd. All rights reserved.