Expression of a Non-Secreted Form of Juvenile Hormone Esterase in a Baculovirus
Pericardial cells rapidly cleared recombinant-juvenile hormone esterase (rJHE) expressed within a baculovirus in insects from the hemolymph. To prevent the clearance of rJHE, we used the polymerase chain reaction (PCR) to remove the signal sequence from the JHE gene, thereby converting the enzyme to a non-secreted form (NSJHE). The resulting gene was expressed in a baculovirus (AcNSJHE) using HIGH-FIVE cells and proper cellular enzyme production was monitored. Transcription level of the NSJHE and rJHE were comparable, and purified NSJHE protein from the cytoplasm hydrolyzed JH. However, efficacy of NSJHE production was low. Enzyme-linked immunosorbent assays and enzyme assays demonstrated enzyme production and activity relative to rJHE of 0.5-1.7% and < 0.1%, respectively. Transmission electron microscopy (TEM) revealed that NSJHE was distributed in the nucleus predominantly and some NSJHE aggregated in clumps within the cytoplasm. These results indicate that NSJHE lacks a specific localization site within cells and that the folding of this enzyme is insufficient. Deglycosylation experiments using purified NSJHE showed that NSJHE was much less glycosylated than rJHE as we expected. Although the NSJHE was less glycosylated, enzyme stability of the NSJHE was equivalent to that of rJHE, indicating that the sugar chains are unimportant in the stability of JHE.
|作成者||TANIAI KiyokoZHOU Carol L. E.LEE Dennis G.MAEDA SusumuHAMMOCK Bruce D.|
|公開者||Japan International Research Center for Agricultural Sciences|
|権利||Japan International Research Center for Agricultural Sciences|