Prorenin processing enzyme (PPE) produced by Baculovirus-infected Sf-9 insect cells: PPE is the cysteine protease encoded in the acMNPV gene.
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.
Gotoh
Takeshi
T
Department of Engineering in Applied Chemistry, Akita University, Japan. tgotoh@ac5.akita-u.ac.jp
Awa
Hirono
H
Kikuchi
Ken-Ichi
K
Nirasawa
Satoru
S
Takahashi
Saori
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000096:biosynthesis*; D057056:Cysteine Proteases / Q000235:genetics*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D018411:Spodoptera / Q000166:cytology* / Q000378:metabolism* / Q000821:virology
D057056:Cysteine Proteases; D003546:Cysteine Endopeptidases; C063281:prorenin processing enzyme