title: Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks.
abstract: Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles.
authors.forename: Michelle
authors.initials: M
authors.lastname: Bradley
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles.
authors.forename: Ivan
authors.initials: I
authors.lastname: Ramirez
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles.
authors.forename: Keith
authors.initials: K
authors.lastname: Cheung
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles.
authors.forename: Ankur A
authors.initials: AA
authors.lastname: Gholkar
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles; Molecular Biology Institute, University of California, Los Angeles; Jonsson Comprehensive Cancer Center, University of California, Los Angeles; torres@chem.ucla.edu.
authors.forename: Jorge Z
authors.initials: JZ
authors.lastname: Torres
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D005822:Genetic Vectors; D006801:Humans; D025941:Protein Interaction Mapping; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods
chemical_list: D011506:Proteins