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Methods Mol Biol. 2018;1728:237-245. doi: 10.1007/978-1-4939-7574-7_15.

Generation of Stable Amber Suppression Cell Lines.

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Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.


Noncanonical amino acid mutagenesis via amber suppression provides the means to tailor proteins inside living cells. A wide range of noncanonical amino acids have been incorporated using the Methanococcus pyrrolysyl-tRNA synthetase/tRNACUA (PylRS/PylT) in mammalian cell systems in proof of principle experiments, for (1) minimal genetically encoded fluorescence or affinity tagging, (2) photo-control of enzymes, (3) genetically encoded posttranslational protein modifications. We have developed a general and efficient method to genomically integrate the PylRS/PylT amber suppression machinery using PiggyBac-mediated transposition. A general protocol for the generation of stable amber suppression cell lines is described here. Using the modular plasmid system, homogenous and highly efficient amber suppression in a wide range of cell lines can be achieved.


Aminoacyl-tRNA-Synthetase; Genetic code expansion; Mammalian cells; Noncanonical amino acids; PiggyBac transposase; Site-specific protein labeling; Unnatural amio acids; tRNA

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