A new method enabling the efficient isolation of protein complexes from mammalian cells is described in an article published online by Nature Methods. The technique can identify the partners in protein interaction networks, known as the 'interactome', which will help researchers to understand how specific proteins interact and function together in the cell.
A widely used affinity purification technique, tandem affinity purification, or TAP, uses specially designed purification tags as hooks to fish out a protein of interest and its interaction partners under gentle, close-to-physiological conditions that preserve binding interactions. The protein's interactome can then be identified with the aid of mass spectrometry. However, the method was originally developed in yeast, and the tag constructs were never optimized for their direct application in mammalian cells.
Giulio Superti-Furga and colleagues now describe a TAP-tag variant optimized for use in mammalian cells. Though they constructed and tested several variants, the purification tag with the most efficient properties consisted of two protein G immunoglobulin-binding units and a streptavidin-binding protein separated by a tobacco etch virus (TEV) protease cleavage site. This tag can be appended recombinantly to a protein of interest and used to isolate the protein and its interaction partners from cells in a two-step procedure.
Although the overall procedure is very similar to the yeast TAP method, the new tags enable the purification of mammalian protein complexes from a relatively small amount of cells and in quantities an order of magnitude higher than by using the original TAP tag. With this method, the authors conclude that "Large-scale approaches to explore the human proteome and cellular machinery should become more feasible."
Giulio Superti-Furga (Research Center for Molecular Medicine, Vienna, Austria)
Abstract available online.
(C) Nature Methods press release.
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