title: Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins.
abstract: Fluorescent fusion proteins are widely used to study protein localization and interaction dynamics in living cells. However, to fully characterize proteins and to understand their function it is crucial to determine biochemical characteristics such as enzymatic activity and binding specificity. Here we demonstrate an easy, reliable and versatile medium/high-throughput method to study biochemical and functional characteristics of fluorescent fusion proteins. Using a new system based on 96-well micro plates comprising an immobilized GFP-binding protein (GFP-mulitTrap), we performed fast and efficient one-step purification of different GFP- and YFP-fusion proteins from crude cell lysate. After immobilization we determined highly reproducible binding ratios of cellular expressed GFP-fusion proteins to histone-tail peptides, DNA or selected RFP-fusion proteins. In particular, we found Cbx1 preferentially binding to di-and trimethylated H3K9 that is abolished by phosphorylation of the adjacent serine. DNA binding assays showed, that the MBD domain of MeCP2 discriminates between fully methylated over unmethylated DNA and protein-protein interactions studies demonstrate, that the PBD domain of Dnmt1 is essential for binding to PCNA. Moreover, using an ELISA-based approach, we detected endogenous PCNA and histone H3 bound at GFP-fusions. In addition, we quantified the level of H3K4me2 on nucleosomes containing different histone variants. In summary, we present an innovative medium/high-throughput approach to analyse binding specificities of fluroescently labeled fusion proteins and to detect endogenous interacting factors in a fast and reliable manner in vitro.
authors.affiliation: Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, Planegg-Martinsried, Munich, Germany. pichler@bio.lmu.de
authors.forename: Garwin
authors.initials: G
authors.lastname: Pichler
authors.forename: Antonia
authors.initials: A
authors.lastname: Jack
authors.forename: Patricia
authors.initials: P
authors.lastname: Wolf
authors.forename: Sandra B
authors.initials: SB
authors.lastname: Hake
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D000090266:Chromobox Protein Homolog 5; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C000715593:CBX1 protein, human; D006657:Histones; D055767:Immobilized Proteins; D008164:Luminescent Proteins; D009707:Nucleosomes; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D000090266:Chromobox Protein Homolog 5; D004247:DNA