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Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels

Slimko, Eric M. and Lester, Henry A. (2003) Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels. Journal of Neuroscience Methods, 124 (1). pp. 75-81. ISSN 0165-0270. https://resolver.caltech.edu/CaltechAUTHORS:20200626-123755093

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Abstract

Organisms use synonymous codons in a highly non-random fashion. These codon usage biases sometimes frustrate attempts to express high levels of exogenous genes in hosts of widely divergent species. The Caenorhabditis elegans GluClα1 and GluClβ genes form a functional glutamate and ivermectin-gated chloride channel when expressed in Xenopus oocytes, but expression is weak in mammalian cells. We have constructed synthetic genes that retain the amino acid sequence of the wild-type GluCl channel proteins, but use codons that are optimal for mammalian cell expression. We have tagged the native and codon-optimized GluCl cDNAs with enhanced yellow fluorescent protein (EYFP, GluClα1 subunit) and enhanced cyan fluorescent protein (EFCP, GluClβ subunit), expressed the channels in E18 rat hippocampal neurons and measured the relative expression levels of the two genes with fluorescence microscopy as well as with electrophysiology. Codon optimization provides a 6- to 9-fold increase in expression, allowing the conclusions that the ivermectin-gated channel has an EC50 of 1.2 nM and a Hill coefficient of 1.9. We also confirm that the Y182F mutation in the codon-optimized β subunit results in a heteromeric channel that retains the response to ivermectin while reducing the response to 100 μM glutamate by 7-fold. The engineered GluCl channel is the first codon-optimized membrane protein expressed in mammalian cells and may be useful for selectively silencing specific neuronal populations in vivo.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/s0165-0270(02)00362-xDOIArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2003 Elsevier. Received 18 October 2002; received in revised form 11 December 2002; accepted 11 December 2002. This work was supported by National Institutes of Health (NS 11756 and MH 49176), by the Plum Foundation and by the William T. Gimbel Discovery fund in Neuroscience. We thank David Anderson, Michael Fanselow and Christof Koch for discussion and Sheri McKinney for help with cultures.
Funders:
Funding AgencyGrant Number
NIHNS 11756
NIHMH 49176
Plum FoundationUNSPECIFIED
William T. Gimbel Discovery FundUNSPECIFIED
Subject Keywords:GluCl; Codon optimization; Fusion proteins; Electrophysiology; Excitability; EYFP; ECFP
Issue or Number:1
Record Number:CaltechAUTHORS:20200626-123755093
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200626-123755093
Official Citation:Eric M. Slimko, Henry A. Lester, Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels, Journal of Neuroscience Methods, Volume 124, Issue 1, 2003, Pages 75-81, ISSN 0165-0270, https://doi.org/10.1016/S0165-0270(02)00362-X.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104091
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:26 Jun 2020 21:29
Last Modified:26 Jun 2020 21:29

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