Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate

BMC Biotechnology

Research Article

A. H. M. Nurun Nabi¹  Tomoyo Tsuyuzaki²  Kazal Boron Biswas²  Ayano Higuma²  Tsutomu Nakagawa²  Satoshi Iwamoto²  Akio Ebihara²  Fumiaki Suzuki²  Satoshi Ohno³  Takashi Yokogawa³  Kazuya Nishikawa³  Akiyoshi Boku-Ikeda³  Ayumi Inayama⁴  Takashi Yamaguchi⁴  Erika Abe⁴  Kaoru Inagaki⁴  Naoya Shibata⁴  Shinji Yamashita⁵ 

[1] Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh;Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, 501-1193, Gifu, Japan;Faculty of Engineering, Gifu University, 1-1 Yanagido, 501-1193, Gifu, Japan;Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, 501-1193, Gifu, Japan;United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, 501-1193, Gifu, Japan;

关键词: Angiotensinogen;    Renin;    Angiotensin;    Hypertension;    Plasma renin concentration;    E. coli;    Recombinant protein production;    Auto-induction;   

DOI  :  10.1186/s12896-016-0265-x

 received in 2015-11-24, accepted in 2016-03-30,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundAngiotensinogen (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.ResultsWhen 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 Km values of both oANG preparations were similar; the kcat value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG.ConclusionsRecombinant 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.

【 授权许可】

CC BY   
© Yamashita et al. 2016