Kwon, Inchan and Wang, Pin and Tirrell, David A. (2006) Design of a Bacterial Host for Site-Specific Incorporation of p-Bromophenylalanine into Recombinant Proteins. Journal of the American Chemical Society, 128 (36). pp. 11778-11783. ISSN 0002-7863. doi:10.1021/ja0626281. https://resolver.caltech.edu/CaltechAUTHORS:20150114-104001262
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Abstract
Introduction of a yeast suppressor tRNA (ytRNA^(Phe)_(CUA)) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) into an Escherichia coli expression host allows in vivo incorporation of phenylalanine analogues into recombinant proteins in response to amber stop codons. However, high-fidelity incorporation of non-natural amino acids is precluded in this system by mischarging of ytRNA^(Phe)_(CUA) with tryptophan (Trp) and lysine (Lys). Here we show that ytRNA^(Phe)_(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF). Two strategies were used to reduce misincorporation of Trp and Lys. First, Lys misincorporation was eliminated by disruption of a Watson−Crick base pair between nucleotides 30 and 40 in ytRNA^(Phe)_(CUA). Loss of this base pair reduces mischarging by the E. coli lysyl-tRNA synthetase. Second, the binding site of yPheRS was redesigned to enhance specificity for pBrF. Specifically, we used the T415A variant, which exhibits 5-fold higher activity toward pBrF as compared to Trp in ATP−PP_i exchange assays. Combining mutant ytRNA^(Phe)_(CUA) and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.
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| Additional Information: | Copyright © 2006 American Chemical Society. Published In Issue September 13, 2006. Received April 14, 2006. We thank Dr. P. Plateau for providing the plasmid pXLysKS1 and Dr. M. Shahgholi for assistance with mass spectrometry. We are grateful to Dr. K. Shin, Dr. J. Petersson, and Dr. D. Beene for help with in vitro transcription of tRNA. We thank I. C. Tanrikulu for help with analysis of the PheRS crystal structure. We also thank Dr. A. J. Link and K. E. Beatty for discussion and valuable comments. This work was supported by National Institutes of Health grant GM62523. | |||||||||
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| Issue or Number: | 36 | |||||||||
| DOI: | 10.1021/ja0626281 | |||||||||
| Record Number: | CaltechAUTHORS:20150114-104001262 | |||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20150114-104001262 | |||||||||
| Official Citation: | Design of a Bacterial Host for Site-Specific Incorporation of p-Bromophenylalanine into Recombinant Proteins Inchan Kwon,Pin Wang, and David A. Tirrell Journal of the American Chemical Society 2006 128 (36), 11778-11783 | |||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
| ID Code: | 53699 | |||||||||
| Collection: | CaltechAUTHORS | |||||||||
| Deposited By: | Katherine Johnson | |||||||||
| Deposited On: | 14 Jan 2015 19:03 | |||||||||
| Last Modified: | 10 Nov 2021 20:05 |
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