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A simple assay for screening translational activity of non-natural amino acids. Implications for polymer synthesis on messenger RNA templates

Kothakota, Srinivas and Yoshikawa, Eiichiro and Murphy, Owen J. and Mason, Thomas L. and Tirrell, David A. and Fournier, Maurille J. (1995) A simple assay for screening translational activity of non-natural amino acids. Implications for polymer synthesis on messenger RNA templates. Journal of Polymer Science Part A: Polymer Chemistry, 33 (8). pp. 1267-1274. ISSN 0887-624X. https://resolver.caltech.edu/CaltechAUTHORS:20150303-161231625

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Abstract

The ability to incorporate unnatural amino acids into biologically synthesized proteins will greatly extend the impact of protein engineering on polymer materials science. The present report describes the use of a rapid cell-free assay to assess the incorporation potential of unnatural amino acids in Escherichia coli. The assay features a coupled transcription-translation system (‘Zubay system’) to screen incorporation of amino acid analogs into plasmid-encoded proteins. Activity estimates are based on the ability of an analog to compete with a radiolabeled natural amino acid, and toxicity effects are screened by monitoring incorporation of a second, unrelated amino acid. The assay was established with analogs known to be active in vivo, using a common bacterial expression vector as template DNA. Positive results were obtained with the leucine analog 5,5,5-trifluoroleucine, the proline analogs azetidine-2-carboxylic acid and thiazolidine-4-carboxylic acid, and three isomers of mono-fluorophenylalanine (o,m,p). No activity was observed for the phenylalanine analogs 2-thienylalanine and 3-thienylalanine. The results suggest that the cell-free assay will be a useful predictor of in vivo incorporation and a useful tool in the design and synthesis of genetically engineered materials.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1002/pola.1995.080330809DOIArticle
http://onlinelibrary.wiley.com/doi/10.1002/pola.1995.080330809/abstractPublisherArticle
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
Additional Information:Copyright © 1995 John Wiley & Sons, Inc. Issue published online: 10 MAR 2003. Article first published online: 10 MAR 2003. Manuscript Accepted: 25 OCT 1994. Manuscript Received: 18 OCT 1994. This work was supported by the NSF Materials Research Laboratory at the University of Massachusetts. E.Y. thanks Kobe Steel Ltd. for a leave of absence to study at the University of Massachusetts. We thank A. Balakin and J. Wower for helpful advice and discussion.
Funders:
Funding AgencyGrant Number
NSF Materials Research LaboratoryUNSPECIFIED
Subject Keywords: unnatural amino acids; 5,5,5-trifluoroleucine; azetidine-2-carboxylic acid; thiazolidine-4-carboxylic acid; mono-fluorophenylalanine (o,m,p); thienylalanine; cellfree protein synthesis
Issue or Number:8
Record Number:CaltechAUTHORS:20150303-161231625
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150303-161231625
Official Citation:Kothakota, S., Yoshikawa, E., Murphy, O. J., Mason, T. L., Tirrell, D. A. and Fournier, M. J. (1995), A simple assay for screening translational activity of non-natural amino acids. Implications for polymer synthesis on messenger RNA templates. J. Polym. Sci. A Polym. Chem., 33: 1267–1274. doi: 10.1002/pola.1995.080330809
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:54562
Collection:CaltechAUTHORS
Deposited By: Anne Hormann
Deposited On:04 Mar 2015 00:21
Last Modified:03 Oct 2019 07:58

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