A Highly Efficient Multifunctional Tandem Affinity Purification Approach Applicable to Diverse Organisms

Abstract

Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) Many biological processes are orchestrated by imposing spatial and temporal regulation on interactions between proteins. Therefore, identification of post-translational modifications and interaction networks among proteins is an essential aspect of modern biology. The tandem affinity purification (TAP) 1 method, originally developed for isolation of native protein complexes in yeast (1), allowed for diverse proteins to be easily purified using a single purification scheme. The TAP method works through expression of a fusion protein consisting of the TAP tag and the bait protein. The TAP tag contains two domains and motifs for tandem affinity purification steps; the rationale being that the use of the second step removes common contaminants that might be specific for the first step. Isolated protein complexes can then be used for biochemical experiments or analyzed by liquid-chromatography-tandem mass-spectrometry (LC-MS/MS). The TAP method has been transferred to mammalian cells; however, the overall recovery of native complexes is low (2). Several modified TAP methods have been developed for mammalian cells, using various combinations of affinity tags. Some tags work well for native protein complexes (2-4), and others for purification under denaturing conditions (5). Other TAP systems incorporate fluorescent proteins to allow for protein localization and to facilitate FACS of individual cells expressing various levels of the tagged protein (6, 7). Each of these purification tags has strengths and weaknesses. Here we describe a fluorescent protein-based multifunctional affinity purification (MAP) tag that incorporates the advantages of multiple purification systems into a single tag. We show that the MAP tag allows protein visualization, rapid selection of cell lines, and efficient affinity purification for all proteins tested under either native or denaturing conditions in mammalian cells, Caenorhabditis elegans, and the fission yeast, Schizosaccharomyces pombe. EXPERIMENTAL PROCEDURESPlasmids-Fluorescent proteins EGFP, sfGFP, mVenus, mCherry, and mKate2 were modified by insertion of the His 8 -SBP-FLAG From the ‡Department

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