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PEDS Advance Access published online on February 16, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzh096
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received August 9, 2004
Revised November 30, 2004
Accepted December 15, 2004

Article

Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells

Erwin Ilegems 1, Horst M. Pick 1, and Horst Vogel 1*

1 Institute of Biomolecular Sciences, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland

* To whom correspondence should be addressed.
Horst Vogel, E-mail: horst.vogel{at}epfl.ch


  Abstract

The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRNA.

Keywords: eukaryotic release factor 1; fluorescent proteins; gene silencing; protein design; suppressor tRNA.
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