Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris
Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD).M...
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| Format: | Article |
| Language: | English |
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Tabriz University of Medical Sciences
2022-05-01
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| Series: | BioImpacts |
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| Online Access: | https://bi.tbzmed.ac.ir/PDF/bi-12-203.pdf |
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| author | Shirin Movaghar Asareh Tahereh Savei Sareh Arjmand Seyed Omid Ranaei Siadat Fataneh Fatemi Mehrab Pourmadadi Javad Shabani Shayeh |
| author_facet | Shirin Movaghar Asareh Tahereh Savei Sareh Arjmand Seyed Omid Ranaei Siadat Fataneh Fatemi Mehrab Pourmadadi Javad Shabani Shayeh |
| author_sort | Shirin Movaghar Asareh |
| collection | DOAJ |
| description | Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD).Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production. |
| first_indexed | 2024-12-12T02:38:53Z |
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| id | doaj.art-7a8aee79e9ad46e8bf08c11481b44109 |
| institution | Directory Open Access Journal |
| issn | 2228-5660 2228-5652 |
| language | English |
| last_indexed | 2024-12-12T02:38:53Z |
| publishDate | 2022-05-01 |
| publisher | Tabriz University of Medical Sciences |
| record_format | Article |
| series | BioImpacts |
| spelling | doaj.art-7a8aee79e9ad46e8bf08c11481b441092022-12-22T00:41:13ZengTabriz University of Medical SciencesBioImpacts2228-56602228-56522022-05-0112320321010.34172/bi.2021.23219bi-23219Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastorisShirin Movaghar Asareh0Tahereh Savei1Sareh Arjmand2Seyed Omid Ranaei Siadat3Fataneh Fatemi4Mehrab Pourmadadi5Javad Shabani Shayeh6Protein Research Center, Shahid Beheshti University, Tehran, IranProtein Research Center, Shahid Beheshti University, Tehran, IranProtein Research Center, Shahid Beheshti University, Tehran, IranProtein Research Center, Shahid Beheshti University, Tehran, IranProtein Research Center, Shahid Beheshti University, Tehran, IranProtein Research Center, Shahid Beheshti University, Tehran, IranProtein Research Center, Shahid Beheshti University, Tehran, IranIntroduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD).Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.https://bi.tbzmed.ac.ir/PDF/bi-12-203.pdfranibizumabfab fragmentvegf-apichia pastorissplit reporteregfp |
| spellingShingle | Shirin Movaghar Asareh Tahereh Savei Sareh Arjmand Seyed Omid Ranaei Siadat Fataneh Fatemi Mehrab Pourmadadi Javad Shabani Shayeh Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris BioImpacts ranibizumab fab fragment vegf-a pichia pastoris split reporter egfp |
| title | Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris |
| title_full | Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris |
| title_fullStr | Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris |
| title_full_unstemmed | Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris |
| title_short | Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris |
| title_sort | expression of functional egfp fused antigen binding fragment of ranibizumab in pichia pastoris |
| topic | ranibizumab fab fragment vegf-a pichia pastoris split reporter egfp |
| url | https://bi.tbzmed.ac.ir/PDF/bi-12-203.pdf |
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