versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins高通量生物化学和功能研究多功能工具箱与荧光融合蛋白.pdfVIP

Versatile Toolbox for High Throughput Biochemical and Functional Studies with Fluorescent Fusion Proteins 1 2 1 2 Garwin Pichler *, Antonia Jack , Patricia Wolf , Sandra B. Hake 1 Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, Planegg-Martinsried, Munich, Germany, 2 Center for Integrated Protein Science Munich at the Adolf-Butenandt Institute, Department of Molecular Biology, Ludwig Maximilians University of Munich, Munich, Germany 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