Purification, cloning, expression and characterization of juvenile hormone esterase (JHE) from Manduca sexta and Tenebrio molitor

Juvenile hormone esterase (JHE) is a selective enzyme that hydrolyzes the methyl ester of juvenile hormone. This enzyme plays an important role in the regulation of metamorphosis in caterpillars, and is implicated in additional roles in development and reproduction in other insects. This laboratory has sought to isolate JHE proteins from new species in order to gain new information of JHE biochemistry through comparative studies between different species and different orders of insects. JHE enzymes from two species of insect were purified, cloned and expressed in vitro. The JHE from Manduca sexta was purified to apparent homogeneity from hemolymph of 5th instar larvae using affinity chromatography. The JHE from Tenebrio molitor was purified from 0–2 day old pupae using affinity chromatography as well. From both of the purified JHE proteins, amino acid sequences were obtained from internal peptides and this information was utilized for the design of degenerate PCR primers. Partial cDNA clones were amplified from both species using mRNA of fat bodies as templates in RT-PCR reactions. The partial cDNA information was subsequently used to design various RACE experiments in order to obtain full-length cDNA sequences. Each cDNA coding sequence was inserted into the genome of the baculovirus, Autographa californica multiply occluded nucleopolyhedrosis virus (AcMNPV), for purposes of in vitro protein expression in insect cell culture. Each cDNA was expressed successfully as a functional JH hydrolyzing enzyme. Both recombinant JHEs were secreted from the insect cells. The JHE enzymes were biochemically characterized and their properties were compared to those of JHEs that were previously reported in the literature. The recombinant JHEs were utilized, along with the recombinant JHE of H. virescens in order to do comparative biochemical studies. These studies revealed both apparent differences and similarities that were shared among the JHEs from 3 different species and 2 orders of insects.