Expression of non-secreted form juvenile hormone esterase in baculovirus

Kiyoko Taniai, Bruce D. Hammock

Research output: Contribution to journalArticle

Abstract

Juvenile hormone esterase (JHK) regulates inset t molting and meiamoiphosiby hydrolysis of (he juvénile hormone Mil). Since disappearance of the ,lli in 1 lie hmnolymph by .HIK result in H depression of insect feeding, t hi-, en/> me is ,\ "real t andidate for an in sec t ici da! agent. Om ,11 tempt to express ,] H K in bar tdovirus was successful in producing high le\e] oi en/yme but the secreted JHK was rapidly eliminated from hemolymph and degraded by peril ardiai cells. In order to produre more stable enzyme, we expressed JHK as a non-secreted cv to-olic form in bac iilnvirus. The signal sequence was removed trom .1111- gene by PCH method and expressed in AcMNPY in SFlM or TN-:!(iN cells, '['he protein production was monitored by the enJ.vine assay and Western blot analysis everyday during four days after infection. Western blot analysis showed that the proteins accumulated without degradation during the Run da\s and the amounts were almost equal with lhat oi wild tvpe JHK whi< li remained in relK. However, the peak activity \vi1h modified .IIIK in rX-:S cells at .t day post infection was extremely low (IS,") pino/min'ml) at in lomparKon with t hat of t he wild type JHK ( !2 n mol /mi n /ml ). F he cytosolic ,1H K u as partially purified by affinity chromatography procedure UM rig ! riflnoromet liyl ketone a a ligand. I he deglvcosylation experiment ot the purified protein showed that the cytosolic JHK was not modified with X linked glyran chain. The specific activity ol the enzyme was equal with wild tvpe JHK. I hesr results indicate that only a tew parcent of expressed JHK in c\tosol have eu/yme acti\ii>.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997

Fingerprint

juvenile hormone esterase
Baculoviridae
Western blotting
Western Blotting
Molting
Hemolymph
Protein Sorting Signals
signal peptide
vines
hemolymph
molting
Insects
Hydrolysis
Assays
Proteins
proteins
Genes
hydrolysis
hormones
Hormones

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Expression of non-secreted form juvenile hormone esterase in baculovirus. / Taniai, Kiyoko; Hammock, Bruce D.

In: FASEB Journal, Vol. 11, No. 9, 1997.

Research output: Contribution to journalArticle

Taniai, Kiyoko ; Hammock, Bruce D. / Expression of non-secreted form juvenile hormone esterase in baculovirus. In: FASEB Journal. 1997 ; Vol. 11, No. 9.
@article{fc352f03a1c64d129bcb147448ffaba0,
title = "Expression of non-secreted form juvenile hormone esterase in baculovirus",
abstract = "Juvenile hormone esterase (JHK) regulates inset t molting and meiamoiphosiby hydrolysis of (he juv{\'e}nile hormone Mil). Since disappearance of the ,lli in 1 lie hmnolymph by .HIK result in H depression of insect feeding, t hi-, en/> me is ,\ {"}real t andidate for an in sec t ici da! agent. Om ,11 tempt to express ,] H K in bar tdovirus was successful in producing high le\e] oi en/yme but the secreted JHK was rapidly eliminated from hemolymph and degraded by peril ardiai cells. In order to produre more stable enzyme, we expressed JHK as a non-secreted cv to-olic form in bac iilnvirus. The signal sequence was removed trom .1111- gene by PCH method and expressed in AcMNPY in SFlM or TN-:!(iN cells, '['he protein production was monitored by the enJ.vine assay and Western blot analysis everyday during four days after infection. Western blot analysis showed that the proteins accumulated without degradation during the Run da\s and the amounts were almost equal with lhat oi wild tvpe JHK whi< li remained in relK. However, the peak activity \vi1h modified .IIIK in rX-:S cells at .t day post infection was extremely low (IS,{"}) pino/min'ml) at in lomparKon with t hat of t he wild type JHK ( !2 n mol /mi n /ml ). F he cytosolic ,1H K u as partially purified by affinity chromatography procedure UM rig ! riflnoromet liyl ketone a a ligand. I he deglvcosylation experiment ot the purified protein showed that the cytosolic JHK was not modified with X linked glyran chain. The specific activity ol the enzyme was equal with wild tvpe JHK. I hesr results indicate that only a tew parcent of expressed JHK in c\tosol have eu/yme acti\ii>.",
author = "Kiyoko Taniai and Hammock, {Bruce D.}",
year = "1997",
language = "English (US)",
volume = "11",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "9",

}

TY - JOUR

T1 - Expression of non-secreted form juvenile hormone esterase in baculovirus

AU - Taniai, Kiyoko

AU - Hammock, Bruce D.

PY - 1997

Y1 - 1997

N2 - Juvenile hormone esterase (JHK) regulates inset t molting and meiamoiphosiby hydrolysis of (he juvénile hormone Mil). Since disappearance of the ,lli in 1 lie hmnolymph by .HIK result in H depression of insect feeding, t hi-, en/> me is ,\ "real t andidate for an in sec t ici da! agent. Om ,11 tempt to express ,] H K in bar tdovirus was successful in producing high le\e] oi en/yme but the secreted JHK was rapidly eliminated from hemolymph and degraded by peril ardiai cells. In order to produre more stable enzyme, we expressed JHK as a non-secreted cv to-olic form in bac iilnvirus. The signal sequence was removed trom .1111- gene by PCH method and expressed in AcMNPY in SFlM or TN-:!(iN cells, '['he protein production was monitored by the enJ.vine assay and Western blot analysis everyday during four days after infection. Western blot analysis showed that the proteins accumulated without degradation during the Run da\s and the amounts were almost equal with lhat oi wild tvpe JHK whi< li remained in relK. However, the peak activity \vi1h modified .IIIK in rX-:S cells at .t day post infection was extremely low (IS,") pino/min'ml) at in lomparKon with t hat of t he wild type JHK ( !2 n mol /mi n /ml ). F he cytosolic ,1H K u as partially purified by affinity chromatography procedure UM rig ! riflnoromet liyl ketone a a ligand. I he deglvcosylation experiment ot the purified protein showed that the cytosolic JHK was not modified with X linked glyran chain. The specific activity ol the enzyme was equal with wild tvpe JHK. I hesr results indicate that only a tew parcent of expressed JHK in c\tosol have eu/yme acti\ii>.

AB - Juvenile hormone esterase (JHK) regulates inset t molting and meiamoiphosiby hydrolysis of (he juvénile hormone Mil). Since disappearance of the ,lli in 1 lie hmnolymph by .HIK result in H depression of insect feeding, t hi-, en/> me is ,\ "real t andidate for an in sec t ici da! agent. Om ,11 tempt to express ,] H K in bar tdovirus was successful in producing high le\e] oi en/yme but the secreted JHK was rapidly eliminated from hemolymph and degraded by peril ardiai cells. In order to produre more stable enzyme, we expressed JHK as a non-secreted cv to-olic form in bac iilnvirus. The signal sequence was removed trom .1111- gene by PCH method and expressed in AcMNPY in SFlM or TN-:!(iN cells, '['he protein production was monitored by the enJ.vine assay and Western blot analysis everyday during four days after infection. Western blot analysis showed that the proteins accumulated without degradation during the Run da\s and the amounts were almost equal with lhat oi wild tvpe JHK whi< li remained in relK. However, the peak activity \vi1h modified .IIIK in rX-:S cells at .t day post infection was extremely low (IS,") pino/min'ml) at in lomparKon with t hat of t he wild type JHK ( !2 n mol /mi n /ml ). F he cytosolic ,1H K u as partially purified by affinity chromatography procedure UM rig ! riflnoromet liyl ketone a a ligand. I he deglvcosylation experiment ot the purified protein showed that the cytosolic JHK was not modified with X linked glyran chain. The specific activity ol the enzyme was equal with wild tvpe JHK. I hesr results indicate that only a tew parcent of expressed JHK in c\tosol have eu/yme acti\ii>.

UR - http://www.scopus.com/inward/record.url?scp=33750192985&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33750192985&partnerID=8YFLogxK

M3 - Article

VL - 11

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 9

ER -