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 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.
Yokogawa
Takashi
T
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan. don@gifu-u.ac.jp
Ohno
Satoshi
S
Nishikawa
Kazuya
K
D000818:Animals; D001386:Azides / Q000378:metabolism*; D002147:Calmodulin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D001386:Azides; D002147:Calmodulin; D005656:Fungal Proteins; D011994:Recombinant Proteins; C065394:3-azidotyrosine; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase