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.
著者
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Gotoh Takeshi
Department of Engineering in Applied Chemistry, Akita University
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Awa Hirono
Department of Materials-Process Engineering and Applied Chemistry for Environments, Akita University
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Kikuchi Ken-Ichi
Department of Engineering in Applied Chemistry, Akita University
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NIRASAWA Satoru
Post-Harvest Science and Technology Division, Japan International Research Center for Agricultural S
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TAKAHASHI Saori
Akita Research Institute of Food and Brewing
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Gotoh Takeshi
Department Of Engineering In Applied Chemistry Graduate School Of Engineering And Resource Science A
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