Identification of a cDNA for SSRP1, an HMG-box protein, by interaction with the c-Myc oncoprotein in a novel bacterial expression screen.
We describe a system for screening cDNA expression libraries in Escherichia coli based on protein-protein interactions. The system utilizes fusion proteins containing the DNA binding domain of the lambda phage cl repressor and a heterologous dimerization domain, which is the target of the screen. Such chimeric proteins were functional as transcriptional repressors in E.coli; function was dependent on the presence of the heterologous dimerization domain, and function of the chimeras was disrupted by expression of excess dimerization domain. A screen was designed to identify factors that could interact with the heterologous dimerization domain and thereby inactivate the chimeric repressor. We used this screen to identify factors that could interact with the basic helix-loop-helix/leucine zipper domains of c-Myc, and isolated the cDNA for a previously characterized HMG domain protein that interacts specifically with c-Myc in this system. This screening method could be used with proteins that have the ability to homo- or heterodimerize.
Bunker
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Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Kingston
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D018257:Helix-Loop-Helix Motifs; D006609:High Mobility Group Proteins / Q000235:genetics*; D016350:Leucine Zippers; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D046911:Macromolecular Substances; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C070454:Ssrp1 protein, mouse; D004247:DNA