Chapter title |
Generation of Stable Amber Suppression Cell Lines
|
---|---|
Chapter number | 15 |
Book title |
Noncanonical Amino Acids
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7574-7_15 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7573-0, 978-1-4939-7574-7
|
Authors |
Simon J. Elsässer, Elsässer, Simon J. |
Abstract |
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. |
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