Identification of a juvenile hormone esterase gene by matching its peptide mass fingerprint with a sequence from the Drosophila genome project

Abstract

Juvenile hormone esterase (JHE, EC 3.1.1.1) from whole Drosophila melanogaster prepupae has previously been purified by selective precipitations, isoelectric focussing and two column chromatography steps. JHE bands from dried silver-stained SDS-PAGE gels of that material were digested with trypsin. The masses of the tryptic digest peptides were determined by MALDI-TOF mass spectrometry. Only one predicted gene product (CG8425) from the D. melanogaster genome matches the JHE tryptic fingerprint with high confidence. This predicted JHE sequence includes features that are conserved among all active members of the serine carboxylesterase multigene family as well as features peculiar to JHEs from other species. Also we show that this JHE can be purified by an alternative method using anion exchange chromotography followed by trifluoromethylketone affinity chromatography. A cDNA encoding this JHE was isolated using 3' and 5' RACE. This sequence is in agreement with the Drosophila genome project's prediction except that the sixth predicted intron is not removed; instead there is a stop codon followed by a polyadenylation signal and a polyA tail.