Cell-Free Protein Production pp 227-242
Incorporation of 3-Azidotyrosine into Proteins Through Engineering Yeast Tyrosyl-tRNA Synthetase and Its Application to Site-Selective Protein Modification
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.