| Title |
Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate
|
|---|---|
| Published in |
BMC Biotechnology, April 2016
|
| DOI | 10.1186/s12896-016-0265-x |
| Pubmed ID | |
| Authors |
Shinji Yamashita, Naoya Shibata, Akiyoshi Boku-Ikeda, Erika Abe, Ayumi Inayama, Takashi Yamaguchi, Ayano Higuma, Kaoru Inagaki, Tomoyo Tsuyuzaki, Satoshi Iwamoto, Satoshi Ohno, Takashi Yokogawa, Kazuya Nishikawa, Kazal Boron Biswas, A. H. M. Nurun Nabi, Tsutomu Nakagawa, Fumiaki Suzuki, Akio Ebihara |
| Abstract |
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. 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. 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. |
X Demographics
The data shown below were collected from the profile of 1 X user who shared this research output. Click here to find out more about how the information was compiled.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 1 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 1 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 22 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 22 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Master | 7 | 32% |
| Researcher | 4 | 18% |
| Student > Ph. D. Student | 2 | 9% |
| Professor | 1 | 5% |
| Lecturer | 1 | 5% |
| Other | 1 | 5% |
| Unknown | 6 | 27% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 5 | 23% |
| Immunology and Microbiology | 4 | 18% |
| Agricultural and Biological Sciences | 2 | 9% |
| Medicine and Dentistry | 2 | 9% |
| Chemical Engineering | 1 | 5% |
| Other | 1 | 5% |
| Unknown | 7 | 32% |
Attention Score in Context
This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 08 April 2016.
All research outputs
#18,450,346
of 22,860,626 outputs
Outputs from BMC Biotechnology
#763
of 935 outputs
Outputs of similar age
#220,499
of 301,014 outputs
Outputs of similar age from BMC Biotechnology
#15
of 22 outputs
Altmetric has tracked 22,860,626 research outputs across all sources so far. This one is in the 11th percentile – i.e., 11% of other outputs scored the same or lower than it.
So far Altmetric has tracked 935 research outputs from this source. They typically receive more attention than average, with a mean Attention Score of 7.8. This one is in the 9th percentile – i.e., 9% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 301,014 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 15th percentile – i.e., 15% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 22 others from the same source and published within six weeks on either side of this one. This one is in the 1st percentile – i.e., 1% of its contemporaries scored the same or lower than it.