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Nucleic Acids Research Advance Access published online on January 31, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm003
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

The gateway pDEST17 expression vector encodes a –1 ribosomal frameshifting sequence

Eric J. Belfield*, Richard K. Hughes, Nicolas Tsesmetzis, Mike J. Naldrett and Rod Casey

John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

*To whom correspondence should be addressed. Tel: +44 (0)1603 450517; Fax: (0)1603 450025; E-mail: eric.belfield{at}bbsrc.ac.uk

Received October 16, 2006. Revised December 22, 2006. Accepted December 23, 2006.

The attB1 site in the Gateway (Invitrogen) bacterial expression vector pDEST17, necessary for in vitro site-specific recombination, contains the sequence AAA-AAA. The sequence A-AAA-AAG within the Escherichia coli dnaX gene is recognized as ‘slippery’ and promotes –1 translational frameshifting. We show here, by expressing in E. coli several plant cDNAs with and without single nucleotide deletions close to the translation initiation codons, that pDEST17 is intrinsically susceptible to –1 ribosomal frameshifting at the sequence C-AAA-AAA. The deletion mutants produce correct-sized, active enzymes with a good correlation between enzyme amount and activity. We demonstrate unambiguously the frameshift through a combination of Edman degradation, MALDI-ToF mass fingerprint analysis of tryptic peptides and MALDI-ToF reflectron in-source decay (rISD) sequencing. The degree of frameshifting depends on the nature of the sequence being expressed and ranged from 25 to 60%. These findings suggest that caution should be exercised when employing pDEST17 for high-level protein expression and that the attB1 site has some potential as a tool for studying –1 frameshifting.


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