Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli.
| Autor: | Jiang HK; Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA.; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.; Chemical Biology & Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan.; Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan., Tharp JM; Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA. jemtharp@iu.edu. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2023; Vol. 2676, pp. 101-116. |
| DOI: | 10.1007/978-1-0716-3251-2_7 |
| Abstrakt: | Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid by Methanocaldococcus jannaschii tyrosyl-tRNA synthetase. Using this initiator tRNA/aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNA Pyl pairs from Methanosarcina mazei and Ca. Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons. (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Databáze: | MEDLINE |
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