期刊
NUCLEIC ACIDS RESEARCH
卷 46, 期 1, 页码 1-10出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkx1156
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资金
- German Research Foundation (DFG) [CO822/2-1, CO822/3-1, MO634/13-1]
- DFG
- Leipzig University
The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D-and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptionalmodifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.
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