A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis
Abstract
We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins- HimD, PduB and PhoP- with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .
- Authors:
-
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Dartmouth College, Hanover, NH (United States)
- Oregon Health and Science Univ., Portland, OR (United States)
- Publication Date:
- Research Org.:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 951817
- Report Number(s):
- PNNL-SA-58873
Journal ID: ISSN 1535-3893; 10904; 10904a; 400412000; TRN: US200913%%139
- DOE Contract Number:
- AC05-76RL01830
- Resource Type:
- Journal Article
- Journal Name:
- Journal of Proteome Research
- Additional Journal Information:
- Journal Volume: 8; Journal Issue: 3; Journal ID: ISSN 1535-3893
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; AFFINITY; CROSS-LINKING; FORMALDEHYDE; IN VIVO; MASS SPECTROSCOPY; PATHOGENESIS; PROTEINS; PURIFICATION; RECOMBINANT DNA; SALMONELLA TYPHIMURIUM; TRANSIENTS; Environmental Molecular Sciences Laboratory
Citation Formats
Chowdhury, Saiful M., Shi, Liang, Yoon, Hyunjin, Ansong, Charles, Rommereim, Leah M., Norbeck, Angela D., Auberry, Kenneth J., Moore, R. J., Adkins, Joshua N., Heffron, Fred, and Smith, Richard D. A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis. United States: N. p., 2009.
Web. doi:10.1021/pr800865d.
Chowdhury, Saiful M., Shi, Liang, Yoon, Hyunjin, Ansong, Charles, Rommereim, Leah M., Norbeck, Angela D., Auberry, Kenneth J., Moore, R. J., Adkins, Joshua N., Heffron, Fred, & Smith, Richard D. A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis. United States. https://doi.org/10.1021/pr800865d
Chowdhury, Saiful M., Shi, Liang, Yoon, Hyunjin, Ansong, Charles, Rommereim, Leah M., Norbeck, Angela D., Auberry, Kenneth J., Moore, R. J., Adkins, Joshua N., Heffron, Fred, and Smith, Richard D. 2009.
"A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis". United States. https://doi.org/10.1021/pr800865d.
@article{osti_951817,
title = {A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis},
author = {Chowdhury, Saiful M. and Shi, Liang and Yoon, Hyunjin and Ansong, Charles and Rommereim, Leah M. and Norbeck, Angela D. and Auberry, Kenneth J. and Moore, R. J. and Adkins, Joshua N. and Heffron, Fred and Smith, Richard D.},
abstractNote = {We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins- HimD, PduB and PhoP- with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .},
doi = {10.1021/pr800865d},
url = {https://www.osti.gov/biblio/951817},
journal = {Journal of Proteome Research},
issn = {1535-3893},
number = 3,
volume = 8,
place = {United States},
year = {Tue Feb 10 00:00:00 EST 2009},
month = {Tue Feb 10 00:00:00 EST 2009}
}