Summary
Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463–468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the λ-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463–468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531–537, 2005).
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Acknowledgments
The authors thank Wenhong Yang, and Xinghua Guo for tagging and purifying the E. coli bait proteins and Shamanta Chandran, Michael Davey, Peter Wong, and Constantine Christopoulos for assisting with mass spectrometry. This work was supported by funds from the Ontario Research and Development Challenge Fund and Genome Canada to A.E. and J.G.
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Babu, M., Butl, G., Pogoutse, O., Li, J., Greenblatt, J.F., Emili, A. (2009). Sequential Peptide Affinity Purification System for the Systematic Isolation and Identification of Protein Complexes from Escherichia coli . In: Reinders, J., Sickmann, A. (eds) Proteomics. Methods in Molecular Biology™, vol 564. Humana Press. https://doi.org/10.1007/978-1-60761-157-8_22
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DOI: https://doi.org/10.1007/978-1-60761-157-8_22
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