The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology
Abstract Background Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-10-01
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| Series: | Biological Research |
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| Online Access: | https://doi.org/10.1186/s40659-022-00400-7 |
| _version_ | 1797991819408048128 |
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| author | Maryam Ehsasatvatan Bahram Baghban Kohnehrouz Ashraf Gholizadeh Hamideh Ofoghi Dariush Shanehbandi |
| author_facet | Maryam Ehsasatvatan Bahram Baghban Kohnehrouz Ashraf Gholizadeh Hamideh Ofoghi Dariush Shanehbandi |
| author_sort | Maryam Ehsasatvatan |
| collection | DOAJ |
| description | Abstract Background Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. Results The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. Conclusion The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes. |
| first_indexed | 2024-04-11T08:58:12Z |
| format | Article |
| id | doaj.art-4420ebf8874f4362a2d00c6d69bd918a |
| institution | Directory Open Access Journal |
| issn | 0717-6287 |
| language | English |
| last_indexed | 2024-04-11T08:58:12Z |
| publishDate | 2022-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Biological Research |
| spelling | doaj.art-4420ebf8874f4362a2d00c6d69bd918a2022-12-22T04:33:08ZengBMCBiological Research0717-62872022-10-0155111810.1186/s40659-022-00400-7The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncologyMaryam Ehsasatvatan0Bahram Baghban Kohnehrouz1Ashraf Gholizadeh2Hamideh Ofoghi3Dariush Shanehbandi4Department of Plant Breeding & Biotechnology, Faculty of Agriculture, University of TabrizDepartment of Plant Breeding & Biotechnology, Faculty of Agriculture, University of TabrizDepartment of Animal Biology, Faculty of Natural Science, University of TabrizDepartment of Biotechnology, Iranian Research Organization for Science and Technology (IROST)Immunology Research Center, Tabriz University of Medical SciencesAbstract Background Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. Results The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. Conclusion The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.https://doi.org/10.1186/s40659-022-00400-7Antibody mimeticsChloroplastDARPinHER2Molecular imagingNicotiana tabacum |
| spellingShingle | Maryam Ehsasatvatan Bahram Baghban Kohnehrouz Ashraf Gholizadeh Hamideh Ofoghi Dariush Shanehbandi The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology Biological Research Antibody mimetics Chloroplast DARPin HER2 Molecular imaging Nicotiana tabacum |
| title | The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology |
| title_full | The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology |
| title_fullStr | The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology |
| title_full_unstemmed | The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology |
| title_short | The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology |
| title_sort | production of the first functional antibody mimetic in higher plants the chloroplast makes the darpin g3 for her2 imaging in oncology |
| topic | Antibody mimetics Chloroplast DARPin HER2 Molecular imaging Nicotiana tabacum |
| url | https://doi.org/10.1186/s40659-022-00400-7 |
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