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Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

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The gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence.

Eric J Belfield¹, Richard K Hughes, Nicolas Tsesmetzis

¹John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. eric.belfield@bbsrc.ac.uk

Nucleic Acids Research

|February 3, 2007

Summary

The Gateway pDEST17 vector can cause -1 ribosomal frameshifting at the attB1 site (AAA-AAA) in E. coli, impacting protein expression. This vector sequence can also be a tool for studying frameshifting events.

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Area of Science:

Molecular Biology
Bacterial Genetics
Protein Expression

Background:

The Gateway pDEST17 vector utilizes the attB1 site for recombination.
The Escherichia coli dnaX gene contains a slippery sequence (A-AAA-AAG) known to induce -1 translational frameshifting.
Ribosomal frameshifting can alter protein synthesis and function.

Purpose of the Study:

To investigate the potential for -1 ribosomal frameshifting within the attB1 site of the pDEST17 vector.
To determine the extent and mechanism of frameshifting induced by the pDEST17 vector's attB1 site.
To evaluate the implications of this frameshifting for high-level protein expression.

Main Methods:

Expression of plant cDNAs with and without nucleotide deletions in E. coli using pDEST17.
Analysis of protein products using Edman degradation.
Mass spectrometry techniques including MALDI-ToF mass fingerprinting and MALDI-ToF reflectron in-source decay (rISD) sequencing.

Main Results:

The pDEST17 vector's attB1 site (C-AAA-AAA) is susceptible to -1 ribosomal frameshifting.
Frameshifting occurred in 25-60% of protein expression, dependent on the expressed sequence.
Deletion mutants produced correctly sized, active enzymes, correlating with enzyme amount and activity.

Conclusions:

Caution is advised when using pDEST17 for high-level protein expression due to inherent frameshifting.
The attB1 site sequence demonstrates potential as a tool for studying -1 frameshifting mechanisms.
Understanding frameshifting is crucial for accurate protein expression and functional studies.