A dual-tagging system for the affinity purification of mammalian protein complexes

Journal Article · Mon Jan 01 04:00:00 EST 2007 · BioTechniques

DOI:https://doi.org/10.2144/000112550· OSTI ID:931033

McDonald, W Hayes ^[1]; Huang, Ying ^[1]; Wu, Jun ^[1]; Liu, Yie ^[2]; Wang, Yisong ^[1]

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1. ORNL
2. National Institute on Aging, Baltimore

One popular method to elucidate protein-protein interactions involves the native co-purification of an affinity tagged protein and its interacting partners, which are subsequently identified through mass spectrometry (MS) (1). Although straightforward, reproducible, and broadly employed, this strategy is hampered by the efficacy of protein recoveries both in terms of sensitivity and specificity. This is especially pertinent to methodologies that employ a single-step of purification, where suboptimal enrichment of the bait protein and its partners over background can lead to masking of their signals. Although improvements to MS instrumentation generally increase peptide detection sensitivities, the problem of specificity, i.e. distinguishing specific from non-specific interacting proteins, remains. Thus ultimately, the limiting factor in the identification of specific interacting proteins lies with the purification itself. An effort to resolve this specificity issue has been made with the introduction of the Tandem Affinity Purification (TAP) tag. This construct consists of an IgG-binding domain and calmodulin binding peptide domain separated by a tobacco etch virus (TEV) protease cleavage site (2). The TAP method was originally developed in yeast and has best demonstrated its utility in the systematic identification of numerous multiprotein complexes in the yeast proteome (3). Although modifications to the original TAP have been successful in examining the protein networks of mammalian cells (4-7), the strategy offers a relatively low yield of bait and specific interacting proteins (8), and the success rate are usually on case-by-case basis. In addition, problems inherent to any protein tagging strategy remain, such as variable exposure of the affinity tag, disruption of the bait protein's ability to fold properly, steric exclusion of interacting partners, and/or ectopic overexpression of the fusion protein, which can lead to complications in both the purification and identification of true interactions.

• Research Organization: Oak Ridge National Laboratory (ORNL)
• Sponsoring Organization: SC USDOE - Office of Science (SC)
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 931033
• Journal Information: BioTechniques, Journal Name: BioTechniques Journal Issue: 3 Vol. 43; ISSN BTNQDO; ISSN 0736-6205
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AFFINITY
CALMODULIN
CLEAVAGE
DETECTION
FIAsH
MASS SPECTROSCOPY
MODIFICATIONS
PEPTIDES
PROTEINS
PURIFICATION
SENSITIVITY
SPECIFICITY
TRF2
dual-tag purification
mass spectrometry
protein complexes