title: Generation and Application of Bioluminescent CD95 Ligand Fusion Proteins.
abstract: The quantitative evaluation of the interaction of soluble CD95L with CD95 is not only important for a detailed understanding of CD95 biology but is also of special relevance for the characterization and development of inhibitors of this interaction. The assembly of a CD95L-CD95 complex capable to recruit intracellular factors not only involves pre-assembly of CD95 molecules in the absence of CD95L but is also modulated by cellular factors such as interaction with the actin cytoskeleton and plasma membrane compartmentation of CD95. Due to these influential variables cell-free methods allow only an inadequate analysis of CD95L binding to cell expressed CD95. To enable easy, sensitive and highly reproducible cellular binding studies for the investigation of the CD95L-CD95 interaction, we generated fusion proteins of soluble CD95L with the luciferase from Gaussia princeps (GpL). The GpL domain contained in the GpL-CD95L fusion proteins does not interfere with CD95 binding and makes the GpL-CD95L fusion proteins highly suitable for cellular binding studies and tracer applications. In this chapter, we report detailed protocols for the production of GpL-CD95L fusion proteins and their use in cellular binding studies.
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070, Würzburg, Germany.
authors.forename: Isabell
authors.initials: I
authors.lastname: Lang
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070, Würzburg, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Kums
authors.affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070, Würzburg, Germany. harald.wajant@mail.uni-wuerzburg.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Wajant
mesh_terms: D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D053222:Fas Ligand Protein / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism
chemical_list: D053222:Fas Ligand Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019014:fas Receptor
keywords: Apoptosis; CD95; CD95L; Gaussia princeps luciferase; Lipid rafts; Tracer