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An engineered Tetrahymena tRNA(Gln) for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression

Saks, Margaret E. and Sampson, Jeffrey R. and Nowak, Mark W. and Kearney, Patrick C. and Du, Fangyong and Abelson, John N. and Lester, Henry A. and Dougherty, Dennis A. (1996) An engineered Tetrahymena tRNA(Gln) for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression. Journal of Biological Chemistry, 271 (38). pp. 23169-23175. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:SAKjbc96

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Abstract

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology.


Item Type:Article
Additional Information:©1996 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, April 1, 1996, and in revised form, June 11, 1996. This research was supported by National Institutes of Health Grants NS11756 (to H.A.L.), NS34407 (to D.A.D.), and GM48560 (to J.N.A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Subject Keywords:TRANSFER RNA-SYNTHETASE; NICOTINIC ACETYLCHOLINE-RECEPTOR; GATED ION CHANNELS; M2 DOMAIN; RECOGNITION; ANTICODON; MUTATIONS; IDENTITY; GENES; ISOPENTENYLATION
Record Number:CaltechAUTHORS:SAKjbc96
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:SAKjbc96
Alternative URL:http://www.jbc.org/cgi/content/abstract/271/38/23169
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4271
Collection:CaltechAUTHORS
Deposited By: Lindsay Cleary
Deposited On:11 Aug 2006
Last Modified:26 Dec 2012 08:58

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