Expression and purification of human (pro)renin receptor in insect cells using baculovirus expression system.
The renin-angiotensin (RA) system is important for the regulation of blood pressure and electrolyte balance, and renin is the rate-limiting enzyme in this system. The recent discovery of (pro)renin receptor (PRR) has reinforced the functional role of the RA system. PRR non-proteolytically activates prorenin and its role has attracted the attention of researchers towards the RA system. However, there is insufficient information on the biochemical structure and biological functioning of PRR due to the difficulty of measuring PRR expression. In this work, human PRR (hPRR) with intact transmembrane and C-terminal domain (hPRR-wTM) and PRR without this domain (hPRR-w/oTM) were expressed in insect cells using baculovirus expression system (BES). Both hPRR-wTM and hPRR-w/oTM were fused with FLAG peptide by its N-terminus. Most of the hPRR-wTM was expressed in cell fraction and hPRR-w/oTM was secreted into the culture medium. hPRR-wTM was solubilized from the membrane fraction of recombinant baculovirus-infected cells by various detergents, suggesting that hPRR-wTM might be a transmembrane protein. hPRR-wTM was purified from the solubilized fraction using anti-FLAG M2 antibody agarose. Binding of purified hPRR-wTM to renin immobilized onto sensor chips was directly proportional to the hPRR-wTM concentration. Approximately 225 microg of functional hPRR-wTM was purified from 80 ml of baculovirus-infected cell culture. Scale-up of this system will lead to mass production and crystallization of hPRR-wTM and determination of its biochemical structure and biological function.
Kato
Tatsuya
T
Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
Kageshima
Ayano
A
Suzuki
Fumiaki
F
Park
Enoch Y
EY
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000302:isolation & purification; D018411:Spodoptera; D020349:Surface Plasmon Resonance; D000093342:Prorenin Receptor
D011956:Receptors, Cell Surface; D000093342:Prorenin Receptor