期刊
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
卷 79, 期 8, 页码 2365-2371出版社
WILEY-BLACKWELL
DOI: 10.1002/prot.23068
关键词
binding artifact; NMR spectroscopy; nucleic acid bridging; misfolding; transcription factors
资金
- National Health and Medical Research Council of Australia (NHMRC)
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 Kruppel-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 mu M. 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.
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