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Site-Specific Incorporation of Unnatural Amino Acids into Proteins in Mammalian Cells

  • Nobumasa Hino
  • Kensaku Sakamoto
  • Shigeyuki YokoyamaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 794)

Abstract

Expanding the repertoire of genetically encoded amino acids in cultured mammalian cells requires the expression of the bacterial or archaeal pair of a tRNA and an aminoacyl-tRNA synthetase variant engineered to be specific to the amino acid, along with the supplementation of an unnatural amino acid in the growth medium. The expression of the pair is generally achieved by transfecting the cultured cells with the plasmids bearing the genes encoding the exogenous pair of translation molecules. Here, we provide a description of some of these plasmids and protocols for transfecting cells with the plasmids and preparing growth media supplemented with unnatural amino acids, to facilitate their incorporation into proteins at specific sites.

Key words

Unnatural amino acids Mammalian cells Genetic code tRNA Aminoacyl-tRNA synthetase OriP Photo-cross-linker Azido group Acetyllysine Iodotyrosine 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Nobumasa Hino
    • 1
  • Kensaku Sakamoto
    • 1
  • Shigeyuki Yokoyama
    • 1
    • 2
    Email author
  1. 1.RIKEN Systems and Structural Biology CenterYokohamaJapan
  2. 2.Department of Biophysics and BiochemistryThe University of TokyoTokyoJapan

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