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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Williams, Thomas L., Iskandar, Debra J., Nödling, Alexander R., Tan, Yurong, Luk, Louis Y. P. and Tsai, Yu-Hsuan 2021. Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency. Amino Acids 53 , pp. 89-96. 10.1007/s00726-020-02927-z

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Abstract

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Additional Information: Open Access This article is licensed under a Creative Commons Attribution 4.0 International License
Publisher: Springer Verlag (Germany)
ISSN: 0939-4451
Funders: Cardiff University
Date of First Compliant Deposit: 4 January 2021
Date of Acceptance: 23 November 2020
Last Modified: 20 May 2021 01:29
URI: http://orca.cardiff.ac.uk/id/eprint/137252

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