Format

Send to

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

Generation of Stable Amber Suppression Cell Lines.

Author information

1
Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. simon.elsasser@scilifelab.se.

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.

KEYWORDS:

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

PMID:
29405002
DOI:
10.1007/978-1-4939-7574-7_15
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center