title: Proteins interacting with cloning scars: a source of false positive protein-protein interactions.
abstract: A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.
authors.affiliation: Stowers Institute for Medical Research, Kansas City, MO 64110.
authors.forename: Charles A S
authors.initials: CA
authors.lastname: Banks
authors.affiliation: Stowers Institute for Medical Research, Kansas City, MO 64110.
authors.forename: Gina
authors.initials: G
authors.lastname: Boanca
authors.affiliation: Stowers Institute for Medical Research, Kansas City, MO 64110.
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Lee
authors.affiliation: Stowers Institute for Medical Research, Kansas City, MO 64110.
authors.forename: Laurence
authors.initials: L
authors.lastname: Florens
authors.affiliation: 1] Stowers Institute for Medical Research, Kansas City, MO 64110 [2] Departments of Pathology &Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Washburn
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D016366:Open Reading Frames / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13 / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D011506:Proteins; D011993:Recombinant Fusion Proteins; C434062:TNIP2 protein, human; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13