In vitro expression and biochemical characterization of juvenile hormone esterase from Manduca sexta.
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 this and other orders of insect. The full length coding region of the JHE cDNA from Manduca sexta was subcloned into the baculovirus AcMNPV genome under the control of the p10 promoter. The recombinant virus demonstrated the expression of high levels of JHE activity when infected into Hi5 cells from Trichoplusia ni. The recombinant protein was partially purified by anion exchange chromatography and its biochemical characterization showed similar features to the wild type protein. The recombinant JHE has an estimated MW of 66500 Da. Some heterogeneity with the enzyme was observed when analyzed by isoelectric focusing, although the peak of JHE activity was observed at pI=6.0. It is highly sensitive to trifluoroketone inhibitors and certain phosphoramidothiolates, while relatively insensitive to other common esterase inhibitors. Incubating the enzyme with various organic solvents and detergents showed that the enzyme is activated at lower concentrations of solvents/detergents and remains significantly active even at high concentrations. The high tolerance of organic solvents may make this JHE enzyme useful in future applications as a synthetic catalyst.
Hinton
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AC
Department of Entomology and Cancer Research Center, University of California, 303 Briggs Hall, 1 Shields Avenue, Davis, CA 95616, USA.
Hammock
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BD
D000818:Animals; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018402:Manduca / Q000201:enzymology*; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase