Cloning and sequencing of a cDNA for the hemolymph juvenile hormone binding protein of larval Manduca sexta.

Abstract

A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been cloned and sequenced. The JHBP was purified to homogeneity from fifth instar larval hemolymph using gel filtration chromatography, ion exchange chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antibodies, generated in response to this protein, were used to identify and isolate JHBP cDNAs from a fat body expression library in bacteriophage lambda ZAPII. Eleven putative JHBP cDNA clones were isolated and subcloned into Bluescript plasmid; cDNA inserts were approximately 750 base pairs in length. A 36-kDa immunoreactive protein was expressed from these plasmids; this beta-galactosidase fusion protein, like the authentic 32-kDa JHBP, was specifically photoaffinity labeled with [3H] epoxyhomofarnesyl diazoacetate (EHDA). Single-stranded DNA from one clone was sequenced by the Sanger dideoxynucleotide method, using deletion and custom primer techniques. A mature translation product was identified which had 226 amino acid residues, a molecular mass of 25,111 daltons, and a predicted isoelectric point (pI) of 5.40. The cDNA correctly predicts the N-terminal amino acid sequence and the amino acid composition of an authentic M. sexta hemolymph JHBP. A computer search of protein and nucleic acid data bases failed to reveal any related sequences. Thus, M. sexta hemolymph JHBP appears to be the first member of a new superfamily of insect hormone binding proteins.