A method for investigating protein-protein interactions related to salmonella typhimurium pathogenesis.
We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). 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 and permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of noncross-linked proteins to bait proteins. Two different negative controls were employed to eliminate the possibility of identifying background and nonspecific proteins as interacting partners, especially those caused by nonspecific binding to the stationary phase used for protein purification. In an initial demonstration of this approach, we tagged three Salmonella proteinsHimD, PduB and PhoPwith known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified for each bait protein, including the known binding partners such as HimA for HimD, as well as unexpected binding partners. Our results suggest that novel protein-protein interactions identified may be critical to pathogenesis by Salmonella.
Chowdhury
Saiful M
SM
Pacific Northwest National Laboratory, Richland, Washington 99352, Oregon Health and Science University, Portland, Oregon 97239, USA.
Shi
Liang
L
Yoon
Hyunjin
H
Ansong
Charles
C
Rommereim
Leah M
LM
Norbeck
Angela D
AD
Auberry
Kenneth J
KJ
Moore
Ronald J
RJ
Adkins
Joshua N
JN
Heffron
Fred
F
Smith
Richard D
RD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology*; D012486:Salmonella typhimurium / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D005557:Formaldehyde; D006639:Histidine