Cell engineering and cultivation of chinese hamster ovary cells for the development of orthogonal eukaryotic cell-free translation systems
- The investigation of protein structures, functions and interactions often requires modifications to adapt protein properties to the specific application. Among many possible methods to equip proteins with new chemical groups, the utilization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs enables the site-specific incorporation of non-canonical amino acids at defined positions in the protein. The open nature of cell-free protein synthesis reactions provides an optimal environment, as the orthogonal components do not need to be transported across the cell membrane and the impact on cell viability is negligible. In the present work, it was shown that the expression of orthogonal aminoacyl-tRNA synthetases in CHO cells prior to cell disruption enhanced the modification of the pharmaceutically relevant adenosine A2a receptor. For this purpose, in complement to transient transfection of CHO cells, an approach based on CRISPR/Cas9 technology was selected to generate a translationally active cell lysate harboring endogenous orthogonalThe investigation of protein structures, functions and interactions often requires modifications to adapt protein properties to the specific application. Among many possible methods to equip proteins with new chemical groups, the utilization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs enables the site-specific incorporation of non-canonical amino acids at defined positions in the protein. The open nature of cell-free protein synthesis reactions provides an optimal environment, as the orthogonal components do not need to be transported across the cell membrane and the impact on cell viability is negligible. In the present work, it was shown that the expression of orthogonal aminoacyl-tRNA synthetases in CHO cells prior to cell disruption enhanced the modification of the pharmaceutically relevant adenosine A2a receptor. For this purpose, in complement to transient transfection of CHO cells, an approach based on CRISPR/Cas9 technology was selected to generate a translationally active cell lysate harboring endogenous orthogonal aminoacyl-tRNA synthetase.…
Verfasserangaben: | Jeffrey Schloßhauer, Niño Cavak, Anne ZemellaORCiDGND, Lena ThoringGND, Stefan KubickORCiD |
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DOI: | https://doi.org/10.3389/fmolb.2022.832379 |
ISSN: | 2296-889X |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/35586195 |
Titel des übergeordneten Werks (Englisch): | Frontiers in molecular biosciences |
Verlag: | Frontiers Media |
Verlagsort: | Lausanne |
Publikationstyp: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Datum der Erstveröffentlichung: | 14.04.2022 |
Erscheinungsjahr: | 2022 |
Datum der Freischaltung: | 06.03.2024 |
Freies Schlagwort / Tag: | C12orf35; CRISPR; E. coli tyrosyl-tRNA synthetase; M. mazei pyrrolysyl-tRNA synthetase; amber suppression; cell-free protein synthesis; membrane protein; orthogonal translation |
Band: | 9 |
Aufsatznummer: | 832379 |
Seitenanzahl: | 13 |
Organisationseinheiten: | Fakultät für Gesundheitswissenschaften |
DDC-Klassifikation: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Peer Review: | Referiert |
Publikationsweg: | Open Access / Gold Open-Access |
DOAJ gelistet | |
Lizenz (Deutsch): | CC-BY - Namensnennung 4.0 International |