Prorenin Processing Enzyme (PPE) Produced by Baculovirus-Infected Sf-9 Insect Cells : PPE Is the Cysteine Protease Encoded in the AcMNPV Gene

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概要

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• In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610–2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.

• 社団法人 日本農芸化学会の論文
• 2010-02-23

著者

• Takahashi S

  Akita Res. Inst. Food And Brewing Akita Jpn

• Gotoh Takeshi

  Department of Engineering in Applied Chemistry, Akita University

• Awa Hirono

  Department of Materials-Process Engineering and Applied Chemistry for Environments, Akita University

• Kikuchi Ken-Ichi

  Department of Engineering in Applied Chemistry, Akita University

• NIRASAWA Satoru

  Post-Harvest Science and Technology Division, Japan International Research Center for Agricultural S

• TAKAHASHI Saori

  Akita Research Institute of Food and Brewing

• Takahashi S

  Kyoto Univ. Kyoto Jpn

• Takahashi S

  Akita Res. Inst. Of Food And Brewing

• Takahashi Saori

  Akita Res. Inst. Of Food And Brewing

• Kikuchi Ken-ichi

  Department Of Engineering In Applied Chemistry Akita University

• Gotoh Takeshi

  Department Of Engineering In Applied Chemistry Akita University

• Gotoh Takeshi

  Department Of Engineering In Applied Chemistry Graduate School Of Engineering And Resource Science A

• Kikuchi Ken-ichi

  Department Of Engineering In Applied Chemistry Graduate School Of Engineering And Resource Science A

• Awa Hirono

  Department Of Materials-process Engineering And Applied Chemistry For Environments Akita University

• Nirasawa Satoru

  Post-harvest Science And Technology Division Japan International Research Center For Agricultural Sc

• Suzuki Takahito

  Biological Laboratory Faculty Of Science Nara Women's University

• Takahashi S

  Department Of Biology The University Of Tokyo

• Gotoh Takeshi

  Department Of Engineering And Applied Chemistry Graduate School Of Engineering And Resource Science

• Takahashi Satoru

  Institute Of Basic Medical Sceinces And Center For Tara

• KIKUCHI KEN-ICHI

  Department of Chemical Engineering for Resources, Akita University

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