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J Proteome Res. 2008 Aug;7(8):3319-28. doi: 10.1021/pr8001832. Epub 2008 Jul 1.

A general system for studying protein-protein interactions in Gram-negative bacteria.

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1
Biosciences Division, Chemical Sciences Division, Computer Science and Mathematics Division, and Physical Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. pelletierda@ornl.gov

Abstract

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

PMID:
18590317
DOI:
10.1021/pr8001832
[Indexed for MEDLINE]
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