Abstract
A key component in determining the functional role of any protein is the elucidation of its binding partners using protein–protein interaction (PPI) data. Here we examine the use of tandem affinity purification (TAP) tagging to study RNA/DNA helicase PPIs in Escherichia coli. The tag, which consists of a calmodulin-binding region, a TEV protease recognition sequence, and an IgG-binding domain, is introduced into E. coli using a λred recombination system. This method prevents the overproduction of the target protein, which could generate false interactions. The interacting proteins are then affinity purified using double affinity purification steps and are seperated by SDS-PAGE followed by mass spectrometry identification. Each protein identified would represent a physical interaction in the cell. These interactions may potentially be mediated by an RNA/DNA template, for which the helicase would likely be needed to disrupt the secondary structures.
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Acknowledgments
This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) and is dedicated to the loving memory of Minoo Rajabian.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Jessulat, M. et al. (2009). In Vivo Investigation of Protein–Protein Interactions for Helicases Using Tandem Affinity Purification. In: Abdelhaleem, M. (eds) Helicases. Methods in Molecular Biology, vol 587. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-355-8_7
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DOI: https://doi.org/10.1007/978-1-60327-355-8_7
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