An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins.
Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.
Howell
Jenika M
JM
Department of Biological Sciences, University of Calgary, Calgary, Canada.
Winstone
Tara L
TL
Coorssen
Jens R
JR
Turner
Raymond J
RJ
D026481:Blotting, Far-Western / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D004340:Drug Contamination / Q000517:prevention & control; D006639:Histidine; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D040081:Protein Array Analysis / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D009942:Organometallic Compounds; C033223:polyhistidine; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D009571:Nitrilotriacetic Acid