title: Discovering protein interactions and characterizing protein function using HaloTag technology.
abstract: Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast. In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1. These examples demonstrate the power of this technology in enabling the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics.
authors.affiliation: Promega Corporation; Danette.Daniels@promega.com.
authors.forename: Danette L
authors.initials: DL
authors.lastname: Daniels
authors.affiliation: Promega Corporation.
authors.forename: Jacqui
authors.initials: J
authors.lastname: Méndez
authors.affiliation: Promega Corporation.
authors.forename: Hélène
authors.initials: H
authors.lastname: Benink
authors.affiliation: Promega Corporation.
authors.forename: Andrew
authors.initials: A
authors.lastname: Niles
authors.affiliation: Promega Corporation.
authors.forename: Nancy
authors.initials: N
authors.lastname: Murphy
authors.affiliation: MS Bioworks LLC.
authors.forename: Michael
authors.initials: M
authors.lastname: Ford
authors.affiliation: MS Bioworks LLC.
authors.forename: Richard
authors.initials: R
authors.lastname: Jones
authors.affiliation: MS Bioworks LLC.
authors.forename: Ravi
authors.initials: R
authors.lastname: Amunugama
authors.affiliation: MS Bioworks LLC.
authors.forename: David
authors.initials: D
authors.lastname: Allen
authors.affiliation: Promega Corporation.
authors.forename: Marjeta
authors.initials: M
authors.lastname: Urh
mesh_terms: D018797:Cell Cycle Proteins; D057809:HEK293 Cells; D006367:HeLa Cells; D056284:Histone Deacetylase 1 / Q000032:analysis / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D046912:Multiprotein Complexes / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D014157:Transcription Factors / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D000092468:Bromodomain Containing Proteins
chemical_list: D018797:Cell Cycle Proteins; D056284:Histone Deacetylase 1; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C110550:HDAC1 protein, human