Expression and in situ processing of human prorenin to active renin in baculovirus-infected Sf-9 insect cell cultures under several infective conditions

• Published in: Biochemical engineering journal Vol. 43; no. 2; pp. 216 - 220
• Main Authors: Gotoh, Takeshi, Awa, Hirono, Kikuchi, Ken-Ichi, Takahashi, Saori
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.02.2009; Elsevier
• Subjects: Baculovirus; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Prorenin; Protease; Renin; Sf-9 insect cell; Sf-9 insect cell; Renin; Baculovirus; Protease; Prorenin; Human; Cell culture; Enzyme; In situ; Insecta; Virus; Infection; Peptidases; Baculoviridae; Arthropoda; Hydrolases; Aspartic endopeptidases; Invertebrata
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2008.10.007

In the baculovirus expression vector system (BEVS), intrinsic proteases concomitantly produced by infected insect cells have been generally regarded as a defect, because they sometimes degrade expressed recombinant proteins and decrease the productivity. The present study successfully used the proteolysis to generate active recombinant human- (rh) renin after the expression of inactive rh-prorenin. Sf-9 insect cells were infected with recombinant baculoviruses having human preprorenin cDNA in the site of polyhedron gene at several MOIs. At any MOIs, rh-prorenin was expressed in a late phase of infective cultures and processed to active rh-renin in a very late phase. The maximum volumetric yield of active rh-renin was obtained at MOIs of 1 and 10 pfu/cell. The protease activity was examined with an internally quenched fluorogenic substrate newly designed for the processing. The generation of rh-renin was coincided with a considerable increase in a protease activity that was classified into the cysteine protease family, and significantly suppressed by supplementing the culture medium with leupeptin, a cysteine protease inhibitor. This suggested that the cysteine protease was responsible to the processing of rh-prorenin to rh-renin.