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Methods Mol Biol. 2010;607:227-42. doi: 10.1007/978-1-60327-331-2_19.

Incorporation of 3-azidotyrosine into proteins through engineering yeast tyrosyl-tRNA synthetase and its application to site-selective protein modification.

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1
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan. don@gifu-u.ac.jp

Abstract

An efficient method for site-selective introduction of 3-azidotyrosine into proteins has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/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.

PMID:
20204861
DOI:
10.1007/978-1-60327-331-2_19
[Indexed for MEDLINE]
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