Prorenin processing enzyme (PPE) produced by baculovirus-infected Sf-9 insect cells: PPE is the cysteine protease encoded in the AcMNPV gene

• Published in: Bioscience, biotechnology, and biochemistry Vol. 74; no. 2; pp. 370 - 374
• Main Authors: Gotoh, T., Akita Univ. (Japan), Awa, H, Kikuchi, K, Nirasawa, S, Takahashi, S
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 2010; Japan Society for Bioscience Biotechnology and Agrochemistry
• Subjects: Animals; Autographa californica; BACULOVIRIDAE; Baculoviridae - genetics; Baculovirus; Biological and medical sciences; Cell Line; Cysteine Endopeptidases - biosynthesis; Cysteine Proteases - genetics; Fundamental and applied biological sciences. Psychology; http://www.fao.org/aos/agrovoc#c_25452; http://www.fao.org/aos/agrovoc#c_34283; http://www.fao.org/aos/agrovoc#c_36924; http://www.fao.org/aos/agrovoc#c_6242; http://www.fao.org/aos/agrovoc#c_9053; Nuclear polyhedrosis virus; Nucleopolyhedrovirus - genetics; ORGANISME TRANSGENIQUE; prorenin; PROTEASAS; PROTEASE; PROTEASES; RENIN; RENINA; RENINE; Sf-9 insect cell; Spodoptera - cytology; Spodoptera - metabolism; Spodoptera - virology; SPODOPTERA FRUGIPERDA; TRANSGENICOS; TRANSGENICS; Cysteine; Enzyme; Insecta; Sf-9 insect cell; Virus; Infection; protease; Peptidases; baculovirus; Renin; Baculoviridae; Gene; Arthropoda; Hydrolases; Aspartic endopeptidases; prorenin; Invertebrata
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.90724

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.