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Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems

Authors
  • Schloßhauer, Jeffrey L.1, 2
  • Cavak, Niño1
  • Zemella, Anne1
  • Thoring, Lena1
  • Kubick, Stefan1, 2, 3
  • 1 Fraunhofer Institute for Cell Therapy and Immunology (IZI), Potsdam , (Germany)
  • 2 Freie Universität Berlin, Berlin , (Germany)
  • 3 The Brandenburg Medical School Theodor Fontane and the University of Potsdam, Potsdam , (Germany)
Type
Published Article
Journal
Frontiers in Molecular Biosciences
Publisher
Frontiers Media SA
Publication Date
Apr 14, 2022
Volume
9
Identifiers
DOI: 10.3389/fmolb.2022.832379
Source
Frontiers
Keywords
Disciplines
  • Molecular Biosciences
  • Original Research
License
Green

Abstract

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 orthogonal aminoacyl-tRNA synthetase.

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