期刊
NATURE METHODS
卷 14, 期 7, 页码 729-+出版社
NATURE PORTFOLIO
DOI: 10.1038/nmeth.4302
关键词
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资金
- Medical Research Council, UK [MC_U105181009, MC_UP_A024_1008]
- BBSRC [BB/M000842/1]
- ERC Advanced Grant (SGCR) [669351]
- EMBO Fellowship [ALTF 297-2015]
- Boehringer Fonds PhD fellowship
- NSF fellowship [1523390]
- BBSRC [BB/M000842/1] Funding Source: UKRI
- MRC [MC_UP_A024_1008, MC_U105181009] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/M000842/1] Funding Source: researchfish
- Medical Research Council [MC_UP_A024_1008, MC_U105181009] Funding Source: researchfish
- Div Of Biological Infrastructure
- Direct For Biological Sciences [1523390] Funding Source: National Science Foundation
- European Research Council (ERC) [669351] Funding Source: European Research Council (ERC)
The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biological function will be accelerated by general methods to biosynthesize defined phosphoproteins. Here we describe a rapid approach for directly discovering aminoacyl-tRNA synthetase-tRNA pairs that selectively incorporate non-natural amino acids into proteins; our method uses parallel positive selections combined with deep sequencing and statistical analysis and enables the direct, scalable discovery of aminoacyl-tRNA synthetase-tRNA pairs with mutually orthogonal substrate specificity. By combining a method to biosynthesize phosphothreonine in cells with this selection approach, we discover a phosphothreonyl-tRNA synthetase-tRNA(CUA) pair and create an entirely biosynthetic route to incorporating phosphothreonine in proteins. We biosynthesize several phosphoproteins and demonstrate phosphoprotein structure determination and synthetic protein kinase activation.
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