Abstract
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 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 for each bait protein, including the known binding partners such as HimAfor HimD, as well as unexpected binding partners. Our results suggest that novel protein-protein interactions identified may be critical to pathogenesis by Salmonella.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1504-1514 |
| Number of pages | 11 |
| Journal | Journal of Proteome Research |
| Volume | 8 |
| Issue number | 3 |
| DOIs | |
| State | Published - Mar 6 2009 |
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Keywords
- Cross-linking
- Formaldehyde
- HBH tag
- In vivo interactions
- Mass spectrometry
ASJC Scopus subject areas
- Biochemistry
- Chemistry(all)
Cite this
A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis. / Chowdhury, Saiful M.; Shi, Liang; Yoon, Hyunjin; Ansong, Charles; Rommereim, Leah M.; Norbeck, Angela D.; Auberry, Kenneth J.; Moore, Ronald J.; Adkins, Joshua N.; Heffron, Fred; Smith, Richard D.
In: Journal of Proteome Research, Vol. 8, No. 3, 06.03.2009, p. 1504-1514.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis
AU - Chowdhury, Saiful M.
AU - Shi, Liang
AU - Yoon, Hyunjin
AU - Ansong, Charles
AU - Rommereim, Leah M.
AU - Norbeck, Angela D.
AU - Auberry, Kenneth J.
AU - Moore, Ronald J.
AU - Adkins, Joshua N.
AU - Heffron, Fred
AU - Smith, Richard D.
PY - 2009/3/6
Y1 - 2009/3/6
N2 - 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 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 for each bait protein, including the known binding partners such as HimAfor HimD, as well as unexpected binding partners. Our results suggest that novel protein-protein interactions identified may be critical to pathogenesis by Salmonella.
AB - 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 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 for each bait protein, including the known binding partners such as HimAfor HimD, as well as unexpected binding partners. Our results suggest that novel protein-protein interactions identified may be critical to pathogenesis by Salmonella.
KW - Cross-linking
KW - Formaldehyde
KW - HBH tag
KW - In vivo interactions
KW - Mass spectrometry
UR - http://www.scopus.com/inward/record.url?scp=65249116382&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=65249116382&partnerID=8YFLogxK
U2 - 10.1021/pr800865d
DO - 10.1021/pr800865d
M3 - Article
VL - 8
SP - 1504
EP - 1514
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 3
ER -