A General System for Studying Protein-Protein Interactions in Gram-Negative Bacteria

Journal Article · Tue Jan 01 04:00:00 EST 2008 · Journal of Proteome Research

DOI:https://doi.org/10.1021/pr8001832· OSTI ID:934802

Pelletier, Dale A ^[1]; Auberry, Deanna L ^[1]; Buchanan, Michelle V ^[1]; Cannon, Bill ^[2]; Daly, Don S. ^[2]; Doktycz, Mitchel John ^[1]; Foote, Linda J ^[1]; Hooker, Brian ^[2]; Kennel, Steve J ^[1]; Lankford, Patricia K ^[1]; Larimer, Frank W ^[1]; Lu, Tse-Yuan S ^[1]; McDonald, W Hayes ^[1]; McKeown, Catherine K ^[1]; Morrell-Falvey, Jennifer L ^[1]; Owens, Elizabeth T ^[1]; Schmoyer, Denise D ^[1]; Shah, Manesh B ^[1]; Wiley, Steven ^[2]; Wang, Yisong ^[1] ; Gilmore, Jason ^[2]

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1. ORNL
2. Pacific Northwest National Laboratory (PNNL)

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.

• Research Organization: Oak Ridge National Laboratory (ORNL)
• Sponsoring Organization: SC USDOE - Office of Science (SC)
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 934802
• Journal Information: Journal of Proteome Research, Journal Name: Journal of Proteome Research Journal Issue: 8 Vol. 7; ISSN 1535-3893; ISSN 1535-3907
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AFFINITY
BACTERIA
BEARINGS
CLONING
CMCS
ESCHERICHIA COLI
GENETICS
IN VIVO
PLASMIDS
PROTEINS
RECOMBINATION
RHODOPSEUDOMONAS
RNA POLYMERASES
VECTORS