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

• Published in: Journal of proteome research Vol. 7; no. 8; pp. 3319 - 3328
• Main Authors: Pelletier, Dale A, Hurst, Gregory B, Foote, Linda J, Lankford, Patricia K, McKeown, Catherine K, Lu, Tse-Yuan, Schmoyer, Denise D, Shah, Manesh B, Hervey, W. Judson, McDonald, W. Hayes, Hooker, Brian S, Cannon, William R, Daly, Don S, Gilmore, Jason M, Wiley, H. Steven, Auberry, Deanna L, Wang, Yisong, Larimer, Frank W, Kennel, Stephen J, Doktycz, Mitchel J, Morrell-Falvey, Jennifer L, Owens, Elizabeth T, Buchanan, Michelle V
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01-08-2008
• Subjects: AFFINITY; Affinity Labels; BACTERIA; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; BEARINGS; CLONING; Cloning, Molecular; CMCS; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; ESCHERICHIA COLI; Escherichia coli - enzymology; Genetic Vectors; GENETICS; Gram-Negative Bacteria - metabolism; IN VIVO; Molecular Probes; PLASMIDS; Protein Interaction Mapping; Protein Subunits - genetics; Protein Subunits - metabolism; PROTEINS; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RECOMBINATION; RHODOPSEUDOMONAS; Rhodopseudomonas - enzymology; RNA POLYMERASES; Shewanella - enzymology; VECTORS; protein interactions; mass spectrometry; protein complexes; RNA polymerase; affinity purification
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr8001832

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.