A general system for studying protein-protein interactions in gram-negative bacteria

Journal Article · Fri Aug 01 04:00:00 EDT 2008 · Journal of Proteome Research, 7(8):3319-3328

DOI:https://doi.org/10.1021/pr8001832· OSTI ID:949113

Pelletier, Dale A; Hurst, G B; Foote, Linda J; Lankford, Patricia K; McKeown, Cathy K; Lu, Tse-Yuan S; Schmoyer, Denise D; Shah, Manesh B; Hervey, IV, W J; McDonald, W Hayes; Hooker, Brian S; Cannon, William R; Daly, Don S; Gilmore, Jason M; Wiley, H S; Auberry, Deanna L; Wang, Yisong; Larimer, Frank; Kennel, S J; Doktycz, M J ; Morrell-Falvey, Jennifer; Owens, Elizabeth T; Buchanan, M V

One of the most promising of the emerging methods for large-scale studies of interactions among proteins is co-isolation of an affinity-tagged protein and its interaction partners, followed by mass spectrometric identification of the co-purifying proteins. We describe a methodology for systematically identifying the proteins that interact with affinity-tagged “bait” proteins expressed from a medium copy plasmid, which are based on a broad host range (pBBR1MCS5) vector backbone that has been modified to incorporate the Gateway DEST plasmid multiple cloning region. This construct was designed to facilitate expression of fusion proteins bearing an affinity tag, across a range of Gram negative bacterial hosts. We demonstrate the performance of 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 from the RNA polymerase complex from these two species compared favorably with those for both plasmid- and chromosomally-encoded affinity-tagged fusion proteins expressed in a model organism, E. coli.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 949113
• Report Number(s): PNNL-SA-58778; KP1501021
• Journal Information: Journal of Proteome Research, 7(8):3319-3328, Journal Name: Journal of Proteome Research, 7(8):3319-3328
• Country of Publication: United States
• Language: English

Related Subjects

COMPLETE GENOME SEQUENCE
COMPLEXES
ESCHERICHIA-COLI K-12
IDENTIFICATION
INTERACTION NETWORKS
RHODOPSEUDOMONAS-PALUSTRIS
RNA-POLYMERASE
SACCHAROMYCES-CEREVISIAE
SHEWANELLA-ONEIDENSIS
SHOTGUN PROTEOMICS