Abstract
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.
