Cell-Free Protein Production pp 227-242

Part of the Methods in Molecular Biology book series (MIMB, volume 607)

Incorporation of 3-Azidotyrosine into Proteins Through Engineering Yeast Tyrosyl-tRNA Synthetase and Its Application to Site-Selective Protein Modification

Protocol

Abstract

An efficient method for site-selective introduction of 3-azidotyrosine into proteins has been developed. This method utilizes the yeast amber suppressor tRNATyr/mutant tyrosyl-tRNA synthetase (Y43G) pair as the carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system. Using rat calmodulin (CaM) as a model protein, we prepared an unnatural CaM molecule carrying a 3-azidotyrosine residue at the predetermined position 80. The synthesized CaM containing 3-azidotyrosine was site-specifically modified via azido group with a fluorescent alkyne derivative by click chemistry. This method will be useful to prepare not only a cross-linkable protein containing 3-azidotyrosine but also a fluorescent protein with a single fluorophore to facilitate the elucidation of molecular mechanisms of protein functions and protein-to-protein networks.

Key words

3-Azidotyrosine In vitro translation system Suppression Tyrosyl-tRNA synthetase Site-selective chemical modification Click chemistry 

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Takashi Yokogawa
    • 1
  • Satoshi Ohno
    • 1
  • Kazuya Nishikawa
    • 1
  1. 1.Department of Biomolecular Science, Faculty of EngineeringGifu UniversityGifuJapan

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