A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Dartmouth College, Hanover, NH (United States)
- Oregon Health and Science Univ., Portland, OR (United States)
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. .
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 951817
- Report Number(s):
- PNNL-SA-58873; 10904; 10904a; 400412000; TRN: US200913%%139
- Journal Information:
- Journal of Proteome Research, Vol. 8, Issue 3; ISSN 1535-3893
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
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