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Experimental Methods Overview

Our pipeline has been developed to identify protein-protein interactions. The endogenous "pulldown" protocol (see flowchart below) is based on a two-stage affinity purification protocol, similar to one that had proven successful in isolating protein complexes from yeast [Gavin, et al, 2002] and E. coli [Butland et al., 2005], but modified for use in Genomic Science microorganisms using our "universal" Gateway-compatible expression vector. We are currently using bait proteins tagged at the C-terminus with both a V5 epitope and a hexahistidine (6xHis) sequence ("tandem tags") [Puig, 2001]. The genes of the targeted proteins are amplified by PCR and inserted into a universal entry vector. From this construct, the gene can be conveniently inserted into low copy number bacterial expression vectors containing an appropriate affinity tag in-frame and inserted into the target cell. Next, cells expressing the affinity tagged gene from a transfected plasmid are grown under specific experimental conditions, disrupted, fractionated, and the complexes separated from the cellular milieu by affinity isolation of the tagged probe protein. Proteins in the isolated complexes are digested with trypsin and the resulting peptides are characterized using a combined liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Proteins are identified using Sequest [Eng et al., 1994].

 
High-throughput PCR amplification and cloning of selected targets into Gateway Entry vectors. 
Recombination of entry clones into low copy Gateway compatible broad host range expression plasmid. 
Expression of bait V5/6xHis fusion protein in R. palustris or S. oneidensis and endogenous protein complex formation. 
IMAC purification followed by V5 antibody purification of bait protein with interactors. 
Elution of specific interactors. 
Trypsin digestion and LC MS/MS identification of interacting proteins. 
Bioinformatic analysis followed by interaction data visualization. 

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References:

  • Butland et al., 2005: Butland, G.; Peregrin-Alvarez, J. M.; Li, J.; Yang, W. H.; Yang, X. C.; Canadien, V.; Starostine, A.; Richards, D.; Beattie, B.; Krogan, N.; Davey, M.; Parkinson, J.; Greenblatt, J.; Emili, A. Nature 2005, 433, 531-37.
  • Eng et al., 1994: Eng, J. K.; McCormack, A. L.; Yates, J. R., III J.Am.Soc.Mass Spectrom. 1994, 5, 976-89.
  • Gavin et al., 2002: Gavin, A.-C.; Bösche, M.; Krause, R.; Grandi, P.; Marzioch, M.; Bauer, A.; Schultz, J.; Rick, J. M.; Michon, A.-M.; Cruciat, C. M.; Remor, M.; Höfert, C.; Schelder, M.; Brajenovic, M.; Ruffner, H.; Merino, A.; Klein, K.; Hudak, M.; Dickson, D.; Rudi, T.; Gnau, V.; Bauch, A.; Bastuck, S.; Huhse, B.; Leutwein, C.; Heurtier, M.-A.; Copley, R. R.; Edelmann, A.; Querfurth, E.; Rybin, V.; Drewes, G.; Raida, M.; Bouwmeester, T.; Bork, P.; Seraphin, B.; Kuster, B.; Neubauer, G.; Superti-Furga, G. Nature 2002, 415, 141-47.
  • Puig, 2001: Puig, O.; Caspary, F.; Rigaut, G.; Rutz, B.; Bouveret, E.; Bragado-Nilsson, E.; Wilm, M.; Seraphin, B. Methods 2001, 24, 218-29.