Kato Tatsuya, Kageshima Ayano, Suzuki Fumiaki, Park Enoch Y
Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
Protein Expr Purif. 2008 Apr;58(2):242-8. doi: 10.1016/j.pep.2007.11.011. Epub 2007 Dec 4.
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.
肾素-血管紧张素（RA）系统对于血压调节和电解质平衡至关重要，肾素是该系统中的限速酶。（前）肾素受体（PRR）的近期发现强化了RA系统的功能作用。PRR可非蛋白水解性激活肾素原，其作用已引起研究人员对RA系统的关注。然而，由于测量PRR表达存在困难，关于PRR的生化结构和生物学功能的信息不足。在这项工作中，使用杆状病毒表达系统（BES）在昆虫细胞中表达了具有完整跨膜和C末端结构域的人PRR（hPRR-wTM）以及没有该结构域的PRR（hPRR-w/oTM）。hPRR-wTM和hPRR-w/oTM的N末端均与FLAG肽融合。大多数hPRR-wTM表达于细胞组分中，而hPRR-w/oTM分泌到培养基中。通过各种去污剂从重组杆状病毒感染细胞的膜组分中溶解hPRR-wTM，这表明hPRR-wTM可能是一种跨膜蛋白。使用抗FLAG M2抗体琼脂糖从溶解组分中纯化hPRR-wTM。纯化的hPRR-wTM与固定在传感器芯片上的肾素的结合与hPRR-wTM浓度成正比。从80 ml杆状病毒感染的细胞培养物中纯化出约225 μg的功能性hPRR-wTM。扩大该系统规模将导致hPRR-wTM的大规模生产和结晶，并确定其生化结构和生物学功能。
