Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems
Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, the utilization of this technology...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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KeAi Communications Co., Ltd.
2019-12-01
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| Series: | Synthetic and Systems Biotechnology |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X19300584 |
| _version_ | 1797205438126620672 |
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| author | Yonatan Chemla Eden Ozer Michael Shaferman Ben Zaad Rambabu Dandela Lital Alfonta |
| author_facet | Yonatan Chemla Eden Ozer Michael Shaferman Ben Zaad Rambabu Dandela Lital Alfonta |
| author_sort | Yonatan Chemla |
| collection | DOAJ |
| description | Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, the utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. Besides, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which could not be achieved in a living cell. Keywords: Cell free system, Genetic code expansion, Thio-lysine, Simplified extract preparation |
| first_indexed | 2024-04-24T08:51:07Z |
| format | Article |
| id | doaj.art-856329b342164fd4a59955cdb76854cc |
| institution | Directory Open Access Journal |
| issn | 2405-805X |
| language | English |
| last_indexed | 2024-04-24T08:51:07Z |
| publishDate | 2019-12-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj.art-856329b342164fd4a59955cdb76854cc2024-04-16T11:59:40ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2019-12-0144189196Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systemsYonatan Chemla0Eden Ozer1Michael Shaferman2Ben Zaad3Rambabu Dandela4Lital Alfonta5Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P.O.Box. 653, Beer-Sheva, 8410501, IsraelDepartment of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P.O.Box. 653, Beer-Sheva, 8410501, IsraelDepartment of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P.O.Box. 653, Beer-Sheva, 8410501, IsraelDepartment of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P.O.Box. 653, Beer-Sheva, 8410501, IsraelInstitute of Chemical Technology-Indian Oil, Odisha campus, IIT Kharagpur extension center, Mouza Samantpuri, Bhubaneswar, 751013, Odisha, IndiaDepartment of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P.O.Box. 653, Beer-Sheva, 8410501, Israel; Corresponding author.Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, the utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. Besides, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which could not be achieved in a living cell. Keywords: Cell free system, Genetic code expansion, Thio-lysine, Simplified extract preparationhttp://www.sciencedirect.com/science/article/pii/S2405805X19300584 |
| spellingShingle | Yonatan Chemla Eden Ozer Michael Shaferman Ben Zaad Rambabu Dandela Lital Alfonta Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems Synthetic and Systems Biotechnology |
| title | Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems |
| title_full | Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems |
| title_fullStr | Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems |
| title_full_unstemmed | Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems |
| title_short | Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems |
| title_sort | simplified methodology for a modular and genetically expanded protein synthesis in cell free systems |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X19300584 |
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