TY  - JOUR
A1  - Schloßhauer, Jeffrey
A1  - Cavak, Niño
A1  - Zemella, Anne
A1  - Thoring, Lena
A1  - Kubick, Stefan
T1  - Cell engineering and cultivation of chinese hamster ovary cells for the development of orthogonal eukaryotic cell-free translation systems
JF  - Frontiers in molecular biosciences
N2  - 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.
KW  - orthogonal translation
KW  - cell-free protein synthesis
KW  - CRISPR
KW  - amber suppression
KW  - E. coli tyrosyl-tRNA synthetase
KW  - M. mazei pyrrolysyl-tRNA synthetase
KW  - membrane protein
KW  - C12orf35
Y1  - 2022
U6  - https://doi.org/10.3389/fmolb.2022.832379
SN  - 2296-889X
VL  - 9
PB  - Frontiers Media
CY  - Lausanne
ER  -