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 regulating the onset of metamorphosis in lepidopteran insects. After affinity chromatography of the hemolymph proteins of Manduca sexta, the pure JHE protein 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 from 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 bp coding region. The deduced protein sequence contains 573 amino acids. (C) 2001 Elsevier Science Ltd. All rights reserved.