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

Hanhui Ma,Janel R Mclean,Lucy Fang-I Chao,Sebastian Mana-Capelli,Murugan Paramasivam,Kirsten A Hagstrom,Kathleen L Gould,Dannel Mccollum

doi:10.1074/mcp.o111.016246

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) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.

Highlights

The TAP method has been transferred to mammalian cells; the overall recovery of native complexes is low (2)
We show that the multifunctional affinity purification (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
Development of the MAP Tag—To optimize the accessibility of affinity tags for protein purification, we inserted a set of tandem affinity tags into the exposed surface loop between ␤8 and ␤9 of GFP-derived fluorescent proteins, a region previously targeted for this purpose (7, 14, 15)

Summary

Introduction

The TAP method has been transferred to mammalian cells; the overall recovery of native complexes is low (2). 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. Protein Purification and Immunoblotting—U2OS cells stably expressing MAP tagged proteins were lysed on ice in lysis buffer (50 mM Tris-HCl, pH 7.5, 125 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40, 5% Glycerol) containing a protease inhibitor mixture (Roche) as specified by the manufacturer.

Results

Conclusion