title: Protein-protein interactions: analysis of a false positive GST pulldown result.
abstract: One of the most common ways to demonstrate a direct protein-protein interaction in vitro is the glutathione-S-transferse (GST)-pulldown. Here we report the detailed characterization of a putative interaction between two transcription factor proteins, GATA-1 and Krüppel-like factor 3 (KLF3/BKLF) that show robust interactions in GST-pulldown experiments. Attempts to map the interaction interface of GATA-1 on KLF3 using a mutagenic screening approach did not yield a contiguous binding face on KLF3, suggesting that the interaction might be non-specific. NMR experiments showed that the proteins do not interact at protein concentrations of 50-100 μM. Rather, the GST tag can cause part of KLF3 to misfold. In addition to misfolding, the fact that both proteins are DNA-binding domains appears to introduce binding artifacts (possibly nucleic acid bridging) that cannot be resolved by the addition of nucleases or ethidium bromide (EtBr). This study emphasizes the need for caution in relying on GST-pulldown results and related methods, without convincing confirmation from different approaches.
authors.affiliation: School of Molecular Bioscience, The University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Sandra
authors.initials: S
authors.lastname: Wissmueller
authors.forename: Josep
authors.initials: J
authors.lastname: Font
authors.forename: Chu Wai
authors.initials: CW
authors.lastname: Liew
authors.forename: Edward
authors.initials: E
authors.lastname: Cram
authors.forename: Thilo
authors.initials: T
authors.lastname: Schroeder
authors.forename: Jeremy
authors.initials: J
authors.lastname: Turner
authors.forename: Merlin
authors.initials: M
authors.lastname: Crossley
authors.forename: Joel P
authors.initials: JP
authors.lastname: Mackay
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Matthews
mesh_terms: D000818:Animals; D050982:GATA1 Transcription Factor / Q000378:metabolism*; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding
chemical_list: D050982:GATA1 Transcription Factor; C529780:Klf3 protein, mouse; D051741:Kruppel-Like Transcription Factors