A versatile lentiviral expression system to identify mammalian protein-protein interactions.
Protein-protein interactions (PPIs) are central to our understanding of protein function, biological processes and signaling pathways. Affinity purification coupled with mass spectrometry (AP-MS) is a powerful approach for detecting PPIs and protein complexes and relies on the purification of bait proteins using bait-specific binding reagents. These binding reagents may recognize bait proteins directly or affinity tags that are fused to bait proteins. A limitation of the latter approach is that expression of affinity tagged baits is largely constrained to engineered or unnatural cell lines, which results in the AP-MS identification of PPIs that may not accurately reflect those seen in nature. Therefore, generating cell lines stably expressing affinity tagged bait proteins in a broad range of cell types and cell lines is important for identifying PPIs that are dependent on different contexts. To facilitate the identification of PPIs across many mammalian cell types, we developed the mammalian affinity purification and lentiviral expression (MAPLE) system. MAPLE uses recombinant lentiviral technology to stably and efficiently express affinity tagged complementary DNA (cDNA) in mammalian cells, including cells that are difficult to transfect and non-dividing cells. The MAPLE vectors contain a versatile affinity (VA) tag for multi-step protein purification schemes and subcellular localization studies. In this methods article, we present a step-by-step overview of the MAPLE system workflow.
Department of Molecular Genetics, University of Toronto, Toronto, Canada.
Anthony B
AB
Mak
Jason
J
Moffat
D000595:Amino Acid Sequence; D001483:Base Sequence; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry
D011993:Recombinant Fusion Proteins