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. 2019 Mar;14(3):e1800125.
doi: 10.1002/biot.201800125. Epub 2018 Jun 11.

A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry

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A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry

Patrik Fladischer et al. Biotechnol J. .

Abstract

The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNACUA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNACUA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity.

Keywords: Desulfitobacterium hafniense; amber suppression; biorthogonal conjugation; genetic code expansion; para-azido-phenylalanine; pyrrolysyl-tRNA synthetase.

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