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

@article{citeulike:2947821, 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 proteinprotein 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.}, address = {Biosciences Division, Chemical Sciences Division, Computer Science and Mathematics Division, and Physical Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, Biological Sciences Division, Computational Sciences and Mathematics Division, Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, and Graduate School of Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830}, author = {Pelletier, Dale A. and Hurst, Gregory B. and Foote, Linda J. and Lankford, Patricia K. and Mckeown, Catherine K. and Lu, Tse-Yuan and Schmoyer, Denise D. and Shah, Manesh B. and Hervey, Judson W. and Mcdonald, Hayes W. and Hooker, Brian S. and Cannon, William R. and Daly, Don S. and Gilmore, Jason M. and Wiley, Steven H. and Auberry, Deanna L. and Wang, Yisong and Larimer, Frank W. and Kennel, Stephen J. and Doktycz, Mitchel J. and Morrell-Falvey, Jennifer L. and Owens, Elizabeth T. and Buchanan, Michelle V. }, citeulike-article-id = {2947821}, doi = {http://dx.doi.org/10.1021/pr8001832}, journal = {J. Proteome Res.}, keywords = {affinity, bacteria, interaction, protein-protein, tag}, month = {July}, posted-at = {2008-07-01 13:24:33}, priority = {2}, title = {A General System for Studying Protein-Protein Interactions in Gram-Negative Bacteria}, url = {http://dx.doi.org/10.1021/pr8001832}, year = {2008} }

TY - JOUR ID - citeulike:2947821 L3 - citeulike-article-id:2947821 TI - A General System for Studying Protein-Protein Interactions in Gram-Negative Bacteria JF - J. Proteome Res. AD - Biosciences Division, Chemical Sciences Division, Computer Science and Mathematics Division, and Physical Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, Biological Sciences Division, Computational Sciences and Mathematics Division, Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, and Graduate School of Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 N2 - 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 proteinprotein 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. KW - affinity KW - bacteria KW - interaction KW - protein-protein KW - tag AU - Pelletier, Dale AU - Hurst, Gregory AU - Foote, Linda AU - Lankford, Patricia AU - Mckeown, Catherine AU - Lu, Tse-Yuan AU - Schmoyer, Denise AU - Shah, Manesh AU - Hervey, Judson AU - Mcdonald, Hayes AU - Hooker, Brian AU - Cannon, William AU - Daly, Don AU - Gilmore, Jason AU - Wiley, Steven AU - Auberry, Deanna AU - Wang, Yisong AU - Larimer, Frank AU - Kennel, Stephen AU - Doktycz, Mitchel AU - Morrell-Falvey, Jennifer AU - Owens, Elizabeth AU - Buchanan, Michelle PY - 2008/07/01/ UR - http://dx.doi.org/10.1021/pr8001832 ER -