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In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system

Nowak, Mark W. and Gallivan, Justin P. and Silverman, Scott K. and Labarca, Cesar G. and Dougherty, Dennis A. and Lester, Henry A. (1998) In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. In: Ion Channels Part B. Methods in Enzymology. No.293. Academic Press , San Diego, CA, pp. 504-529. ISBN 978-0-12-182194-4. https://resolver.caltech.edu/CaltechAUTHORS:20200515-134619162

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Abstract

This chapter has adapted the nonsense codon suppression method for the incorporation of unnatural amino acids into membrane proteins in a Xenopus oocyte expression system. Combining this method with electrophysiologic analysis allows it to probe structure-function relationships in ion channels and receptors in ways not possible with conventional mutagenesis. In the absence of atomic-scale, structural data for membrane proteins, these techniques can provide detailed structural information. In the nonsense codon suppression method, Xenopus oocytes are coinjected with two RNAs: (1) mRNA transcribed in vitro from a mutated cDNA containing a TAG nonsense (stop) codon at the position of interest and (2) a suppressor tRNA containing the corresponding anticodon, CUA, and chemically acylated at the end with an amino acid. During protein synthesis, the aminoacylated suppressor tRNA directs the incorporation of the amino acid into the desired position of the protein. Because the amino acid is appended synthetically, it is not limited to the natural, and many unnatural amino acids have been incorporated into various proteins using this method. This chapter describes suppressor tRNA design and synthesis, chemical acylation of the suppressor tRNA, relevant organic synthesis methods, and optimization of mRNA and suppressor tRNA for Xenopus oocyte expression.


Item Type:Book Section
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/s0076-6879(98)93031-2DOIArticle
ORCID:
AuthorORCID
Dougherty, Dennis A.0000-0003-1464-2461
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 1998 Published by Elsevier Inc. We thank the NIH (NS-11756 and NS-34407) for support of this work.
Funders:
Funding AgencyGrant Number
NIHNS-11756
NIHNS-34407
Series Name:Methods in Enzymology
Issue or Number:293
Record Number:CaltechAUTHORS:20200515-134619162
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200515-134619162
Official Citation:Mark W. Nowak, Justin P. Gallivan, Scott K. Silverman, Cesar G. Labarca, Dennis A. Dougherty, Henry A. Lester, In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system, Methods in Enzymology, Academic Press, Volume 293, 1998, Pages 504-529, ISSN 0076-6879, ISBN 9780121821944, https://doi.org/10.1016/S0076-6879(98)93031-2. (http://www.sciencedirect.com/science/article/pii/S0076687998930312)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103242
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 May 2020 20:58
Last Modified:15 May 2020 20:58

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