Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System
The spike protein (SP) of SARS-CoV-2 is an important target for COVID-19 therapeutics and vaccines as it binds to the ACE2 receptor and enables viral infection. Rapid production and functional characterization of properly folded SP is of the utmost importance for studying the immunogenicity and rece...
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| Format: | Article |
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MDPI AG
2022-07-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/14/7/1461 |
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| author | Yutaro Yamaoka Sundararaj Stanleyraj Jeremiah Rikako Funabashi Kei Miyakawa Takeshi Morita Yusaku Mihana Hideaki Kato Akihide Ryo |
| author_facet | Yutaro Yamaoka Sundararaj Stanleyraj Jeremiah Rikako Funabashi Kei Miyakawa Takeshi Morita Yusaku Mihana Hideaki Kato Akihide Ryo |
| author_sort | Yutaro Yamaoka |
| collection | DOAJ |
| description | The spike protein (SP) of SARS-CoV-2 is an important target for COVID-19 therapeutics and vaccines as it binds to the ACE2 receptor and enables viral infection. Rapid production and functional characterization of properly folded SP is of the utmost importance for studying the immunogenicity and receptor-binding activity of this protein considering the emergence of highly infectious viral variants. In this study, we attempted to express the receptor-binding region (RBD) of SARS-CoV-2 SP containing disulfide bonds using the wheat germ cell-free protein synthesis system. By adding protein disulfide isomerase (PDI) and endoplasmic reticulum oxidase (ERO1α) to the translational reaction mixture, we succeeded in synthesizing a functionally intact RBD protein that can interact with ACE2. Using this RBD protein, we have developed a high-throughput AlphaScreen assay to evaluate the RBD–ACE2 interaction, which can be applied for drug screening and mutation analysis. Thus, our method sheds new light on the structural and functional properties of SARS-CoV-2 SP and has the potential to contribute to the development of new COVID-19 therapeutics. |
| first_indexed | 2024-03-09T05:43:14Z |
| format | Article |
| id | doaj.art-7c4003c4cf0c41999e91569d20958d5e |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-09T05:43:14Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-7c4003c4cf0c41999e91569d20958d5e2023-12-03T12:23:13ZengMDPI AGViruses1999-49152022-07-01147146110.3390/v14071461Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production SystemYutaro Yamaoka0Sundararaj Stanleyraj Jeremiah1Rikako Funabashi2Kei Miyakawa3Takeshi Morita4Yusaku Mihana5Hideaki Kato6Akihide Ryo7Department of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, JapanDepartment of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, JapanDepartment of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, JapanDepartment of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, JapanDepartment of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, JapanDepartment of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, JapanInfection Prevention and Control Department, Yokohama City University Hospital, Yokohama 236-0004, JapanDepartment of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, JapanThe spike protein (SP) of SARS-CoV-2 is an important target for COVID-19 therapeutics and vaccines as it binds to the ACE2 receptor and enables viral infection. Rapid production and functional characterization of properly folded SP is of the utmost importance for studying the immunogenicity and receptor-binding activity of this protein considering the emergence of highly infectious viral variants. In this study, we attempted to express the receptor-binding region (RBD) of SARS-CoV-2 SP containing disulfide bonds using the wheat germ cell-free protein synthesis system. By adding protein disulfide isomerase (PDI) and endoplasmic reticulum oxidase (ERO1α) to the translational reaction mixture, we succeeded in synthesizing a functionally intact RBD protein that can interact with ACE2. Using this RBD protein, we have developed a high-throughput AlphaScreen assay to evaluate the RBD–ACE2 interaction, which can be applied for drug screening and mutation analysis. Thus, our method sheds new light on the structural and functional properties of SARS-CoV-2 SP and has the potential to contribute to the development of new COVID-19 therapeutics.https://www.mdpi.com/1999-4915/14/7/1461COVID-19SARS-CoV-2RBDdisulfide bondcell-free protein synthesisdrug screening |
| spellingShingle | Yutaro Yamaoka Sundararaj Stanleyraj Jeremiah Rikako Funabashi Kei Miyakawa Takeshi Morita Yusaku Mihana Hideaki Kato Akihide Ryo Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System Viruses COVID-19 SARS-CoV-2 RBD disulfide bond cell-free protein synthesis drug screening |
| title | Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System |
| title_full | Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System |
| title_fullStr | Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System |
| title_full_unstemmed | Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System |
| title_short | Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System |
| title_sort | characterization and utilization of disulfide bonded sars cov 2 receptor binding domain of spike protein synthesized by wheat germ cell free production system |
| topic | COVID-19 SARS-CoV-2 RBD disulfide bond cell-free protein synthesis drug screening |
| url | https://www.mdpi.com/1999-4915/14/7/1461 |
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