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

Dale A. Pelletier, Gregory B. Hurst, Linda J. Foote, Patricia K. Lankford, Catherine K. McKeown, Tse Yuan Lu, Denise D. Schmoyer, Manesh B. Shah, W. Judson Hervey IV, W. Hayes McDonald, Brian S. Hooker, William R. Cannon, Don S. Daly, Jason M. Gilmore, H. Steven Wiley, Deanna L. Auberry, Yisong Wang, Frank W. Larimer, Stephen Kennel, Mitchel J. Doktycz & 3 others Jennifer L. Morrell-Falvey, Elizabeth T. Owens, Michelle V. Buchanan

Research output: Contribution to journalArticle

16 Citations (Scopus)

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, Rhodopseudomo- nas 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.

Original languageEnglish (US)
Pages (from-to)3319-3328
Number of pages10
JournalJournal of Proteome Research
Volume7
Issue number8
DOIs
StatePublished - Aug 1 2008

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Gram-Negative Bacteria
Bacteria
Proteins
Cloning
Organism Cloning
Plasmids
Bearings (structural)
Fusion reactions
Rhodopseudomonas
Shewanella
Host Specificity
DNA-Directed RNA Polymerases
Escherichia coli
Genetic Recombination

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Chemistry(all)

Cite this

Pelletier, D. A., Hurst, G. B., Foote, L. J., Lankford, P. K., McKeown, C. K., Lu, T. Y., ... Buchanan, M. V. (2008). A general system for studying protein-protein interactions in gram-negative bacteria. Journal of Proteome Research, 7(8), 3319-3328. https://doi.org/10.1021/pr8001832

A general system for studying protein-protein interactions in gram-negative bacteria. / 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 IV, 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; Doktycz, Mitchel J.; Morrell-Falvey, Jennifer L.; Owens, Elizabeth T.; Buchanan, Michelle V.

In: Journal of Proteome Research, Vol. 7, No. 8, 01.08.2008, p. 3319-3328.

Research output: Contribution to journalArticle

Pelletier, DA, Hurst, GB, Foote, LJ, Lankford, PK, McKeown, CK, Lu, TY, Schmoyer, DD, Shah, MB, Hervey IV, WJ, McDonald, WH, Hooker, BS, Cannon, WR, Daly, DS, Gilmore, JM, Wiley, HS, Auberry, DL, Wang, Y, Larimer, FW, Kennel, S, Doktycz, MJ, Morrell-Falvey, JL, Owens, ET & Buchanan, MV 2008, 'A general system for studying protein-protein interactions in gram-negative bacteria', Journal of Proteome Research, vol. 7, no. 8, pp. 3319-3328. https://doi.org/10.1021/pr8001832
Pelletier DA, Hurst GB, Foote LJ, Lankford PK, McKeown CK, Lu TY et al. A general system for studying protein-protein interactions in gram-negative bacteria. Journal of Proteome Research. 2008 Aug 1;7(8):3319-3328. https://doi.org/10.1021/pr8001832
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 IV, 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 ; Doktycz, Mitchel J. ; Morrell-Falvey, Jennifer L. ; Owens, Elizabeth T. ; Buchanan, Michelle V. / A general system for studying protein-protein interactions in gram-negative bacteria. In: Journal of Proteome Research. 2008 ; Vol. 7, No. 8. pp. 3319-3328.
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