title: Protein Chemical Modification Inside Living Cells Using Split Inteins.
abstract: Methods to visualize, track, measure, and perturb or activate proteins in living cells are central to biomedical efforts to characterize and understand the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chemical labeling of proteins. We describe in this protocol the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split-inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. We have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, we have shown that this approach can be also used for modifying proteins in order to control their cellular localization and potentially alter their biological activity.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, 90089-9121, USA.
authors.forename: Radhika
authors.initials: R
authors.lastname: Borra
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, 90089-9121, USA. jcamarer@usc.edu.|Department of Chemistry, University of Southern California, Los Angeles, CA, 90089-9121, USA. jcamarer@usc.edu.
authors.forename: Julio A
authors.initials: JA
authors.lastname: Camarero
mesh_terms: D045744:Cell Line, Tumor; D004258:DNA Polymerase III / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047668:Inteins; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III
keywords: Fluorescence; Npu intein; Protein labeling; Protein trans-splicing; Split-intein