期刊
CHEMBIOCHEM
卷 17, 期 16, 页码 1518-1524出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201600284
关键词
amino acids; click chemistry; fluorescence; hydrophilicity; protein engineering
资金
- Hungarian Scientific Research Fund (OTKA) [NN-116265]
- Lendulet Program of the Hungarian Academy of Sciences [LP2013-55/2013]
- FEBS Short-term fellowship
- Campus Hungary
- European Commission
- European Molecular Biology Organization (EMBO)
- Deutsche Forschungsgemeinschaft [SFB1129]
- BMBF (switch click consortium)
Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.
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