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Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate
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...
Autores principales: | , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823841/ https://www.ncbi.nlm.nih.gov/pubmed/27052373 http://dx.doi.org/10.1186/s12896-016-0265-x |
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author | Yamashita, Shinji Shibata, Naoya Boku-Ikeda, Akiyoshi Abe, Erika Inayama, Ayumi Yamaguchi, Takashi Higuma, Ayano Inagaki, Kaoru Tsuyuzaki, Tomoyo Iwamoto, Satoshi Ohno, Satoshi Yokogawa, Takashi Nishikawa, Kazuya Biswas, Kazal Boron Nabi, A. H. M. Nurun Nakagawa, Tsutomu Suzuki, Fumiaki Ebihara, Akio |
author_facet | Yamashita, Shinji Shibata, Naoya Boku-Ikeda, Akiyoshi Abe, Erika Inayama, Ayumi Yamaguchi, Takashi Higuma, Ayano Inagaki, Kaoru Tsuyuzaki, Tomoyo Iwamoto, Satoshi Ohno, Satoshi Yokogawa, Takashi Nishikawa, Kazuya Biswas, Kazal Boron Nabi, A. H. M. Nurun Nakagawa, Tsutomu Suzuki, Fumiaki Ebihara, Akio |
author_sort | Yamashita, Shinji |
collection | PubMed |
description | 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-016-0265-x) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4823841 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-48238412016-04-08 Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate Yamashita, Shinji Shibata, Naoya Boku-Ikeda, Akiyoshi Abe, Erika Inayama, Ayumi Yamaguchi, Takashi Higuma, Ayano Inagaki, Kaoru Tsuyuzaki, Tomoyo Iwamoto, Satoshi Ohno, Satoshi Yokogawa, Takashi Nishikawa, Kazuya Biswas, Kazal Boron Nabi, A. H. M. Nurun Nakagawa, Tsutomu Suzuki, Fumiaki Ebihara, Akio BMC Biotechnol Research Article 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-016-0265-x) contains supplementary material, which is available to authorized users. BioMed Central 2016-04-07 /pmc/articles/PMC4823841/ /pubmed/27052373 http://dx.doi.org/10.1186/s12896-016-0265-x Text en © Yamashita et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Yamashita, Shinji Shibata, Naoya Boku-Ikeda, Akiyoshi Abe, Erika Inayama, Ayumi Yamaguchi, Takashi Higuma, Ayano Inagaki, Kaoru Tsuyuzaki, Tomoyo Iwamoto, Satoshi Ohno, Satoshi Yokogawa, Takashi Nishikawa, Kazuya Biswas, Kazal Boron Nabi, A. H. M. Nurun Nakagawa, Tsutomu Suzuki, Fumiaki Ebihara, Akio Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate |
title | Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate |
title_full | Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate |
title_fullStr | Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate |
title_full_unstemmed | Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate |
title_short | Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate |
title_sort | escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823841/ https://www.ncbi.nlm.nih.gov/pubmed/27052373 http://dx.doi.org/10.1186/s12896-016-0265-x |
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