Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta

Juvenile hormone esterase (JHE) is a highly specific enzyme important for r egulating the onset of metamorphosis in lepidopteran insects. After affinit y chromatography of the hemolymph proteins of Manduca sexta, the pure JHE p rotein was digested with Lys-C and the resultant peptides were purified by microbore HPLC. Two peptides were selected for sequencing. Based upon these amino acid sequences, degenerate RT-PCR was performed in order to amplify a partial cDNA sequence from mRNA from the fat body of M. sexta, A 1512 bp partial cDNA was generated and found to be highly homologous to the JHE fro m Heliothis virescens. 5 ' and 3 ' RACE were per-formed to obtain the full length cDNA sequence. The cDNA has a total length of 2220 bp, with a 1749 b p coding region. The deduced protein sequence contains 573 amino acids. (C) 2001 Elsevier Science Ltd. All rights reserved.