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Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

1
Centre for Mechanochemical Cell Biology and Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Gibbet Hill Road, CV4 7AL Coventry, UK
2
Proteomics Research Technology Platform and School of Life Sciences, University of Warwick, Gibbet Hill Road, CV4 7AL Coventry, UK
*
Author to whom correspondence should be addressed.
These authors contributed equally.
Present address: Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK.
Int. J. Mol. Sci. 2019, 20(10), 2577; https://doi.org/10.3390/ijms20102577
Received: 9 May 2019 / Revised: 22 May 2019 / Accepted: 23 May 2019 / Published: 26 May 2019
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Abstract

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish. View Full-Text
Keywords: genetic code expansion; zebrafish; unnatural amino acid; protein engineering; proteomics; crosslinking; click chemistry genetic code expansion; zebrafish; unnatural amino acid; protein engineering; proteomics; crosslinking; click chemistry
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Syed, J.; Palani, S.; Clarke, S.T.; Asad, Z.; Bottrill, A.R.; Jones, A.M.; Sampath, K.; Balasubramanian, M.K. Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine. Int. J. Mol. Sci. 2019, 20, 2577.

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