title: Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate.
abstract: BACKGROUND
  Angiotensinogen (ANG) is a macromolecular precursor of angiotensin, which regulates blood pressure and electrolyte balance. ANG is specifically cleaved by renin, an aspartic protease, to initiate the angiotensin-processing cascade. Ovine ANG (oANG) from sheep plasma has been shown to be a better substrate for human renin, and it has been used in clinical renin assays. To expand the availability of oANG, we aimed to produce milligram levels of recombinant oANG using an Escherichia coli expression system.
  
  
  RESULTS
  When recombinant oANG was expressed from a T7 promoter in various E. coli strains at 37 °C, it accumulated in the insoluble fraction. However, by expressing oANG at 37 °C from a tac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. Using a novel culturing system and auto-induction culture medium, we purified tac-expressed recombinant oANG to homogeneity, with a yield of 4.0 mg per liter of culture. Based on size-exclusion gel filtration analysis and dynamic light scattering analysis, the resulting purified oANG is a monomer in solution. The circular dichroism spectrum of E. coli-expressed recombinant oANG was similar to that of oANG expressed in CHO cells. Differential scanning fluorimetry showed that both preparations undergo a two-state transition during thermal denaturation, and the melting temperatures of recombinant oANG expressed in E. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively. The K(m) values of both oANG preparations were similar; the k(cat) value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG.
  
  
  CONCLUSIONS
  Recombinant oANG expressed in E. coli functions as a human renin substrate. This study presents an E. coli-based system for the rapid production of milligram quantities of a human renin substrate, which will be useful for both fundamental and clinical studies on renin and hypertension.
authors.affiliation: United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Shibata
authors.affiliation: Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Boku-Ikeda
authors.affiliation: Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Erika
authors.initials: E
authors.lastname: Abe
authors.affiliation: Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Inayama
authors.affiliation: Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Ayano
authors.initials: A
authors.lastname: Higuma
authors.affiliation: Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Inagaki
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Tomoyo
authors.initials: T
authors.lastname: Tsuyuzaki
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Iwamoto
authors.affiliation: Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ohno
authors.affiliation: Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokogawa
authors.affiliation: Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nishikawa
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Kazal Boron
authors.initials: KB
authors.lastname: Biswas
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh.
authors.forename: A H M Nurun
authors.initials: AH
authors.lastname: Nabi
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Suzuki
authors.affiliation: Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. aebihara@gifu-u.ac.jp.
authors.forename: Akio
authors.initials: A
authors.lastname: Ebihara
mesh_terms: D000808:Angiotensinogen / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012083:Renin / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep
chemical_list: D011994:Recombinant Proteins; D000808:Angiotensinogen; D012083:Renin
keywords: Angiotensin; Angiotensinogen; Auto-induction; E. coli; Hypertension; Plasma renin concentration; Recombinant protein production; Renin