CD95 pp 63-77 | Cite as

Generation and Application of Bioluminescent CD95 Ligand Fusion Proteins

  • Isabell Lang
  • Juliane Kums
  • Harald Wajant
Part of the Methods in Molecular Biology book series (MIMB, volume 1557)


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.

Key words

Apoptosis CD95 CD95L Gaussia princeps luciferase Lipid rafts Tracer 


  1. 1.
    Lang I, Füllsack S, Wyzgol A, Fick A, Trebing J, Schäfer V et al (2016) Binding studies of TNF receptor superfamily (TNFRSF) receptors on intact cells. J Biol Chem 291(10):5022–5037CrossRefPubMedGoogle Scholar
  2. 2.
    Wajant H (2014) Principles and mechanisms of CD95 activation. Biol Chem 395:1401–1416CrossRefPubMedGoogle Scholar
  3. 3.
    Wajant H, Gerspach J, Pfizenmaier K (2013) Engineering death receptor ligands for cancer therapy. Cancer Lett 332:163–174CrossRefPubMedGoogle Scholar
  4. 4.
    Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11:435–443CrossRefPubMedGoogle Scholar
  5. 5.
    Schneider P, Holler N, Bodmer JL, Hahne M, Frei K, Fontana A et al (1998) Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J Exp Med 187:1205–1213CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Holler N, Tardivel A, Kovacsovics-Bankowski M, Hertig S, Gaide O, Martinon F et al (2003) Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol Cell Biol 23:1428–1440CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Berg D, Lehne M, Müller N, Siegmund D, Münkel S, Sebald W et al (2007) Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L. Cell Death Differ 14:2021–2034CrossRefPubMedGoogle Scholar
  8. 8.
    Wyzgol A, Müller N, Fick A, Munkel S, Grigoleit GU, Pfizenmaier K et al (2009) Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand. J Immunol 183:1851–1861CrossRefPubMedGoogle Scholar
  9. 9.
    Lang I, Fick A, Schäfer V, Giner T, Siegmund D, Wajant H (2012) Signaling active CD95 receptor molecules trigger co-translocation of inactive CD95 molecules into lipid rafts. J Biol Chem 287:24026–24042CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Rauert H, Stühmer T, Bargou R, Wajant H, Siegmund D (2011) TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms. Cell Death Dis 2:e194CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Fick A, Lang I, Schäfer V, Seher A, Trebing J, Weisenberger D et al (2012) Studies of binding of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to fibroblast growth factor inducible 14 (Fn14). J Biol Chem 287:484–495CrossRefPubMedGoogle Scholar
  12. 12.
    Salzmann S, Lang I, Rosenthal A, Schäfer V, Weisenberger D, Carmona Arana JA et al (2013) TWEAK inhibits TRAF2-mediated CD40 signaling by destabilization of CD40 signaling complexes. J Immunol 191:2308–2318CrossRefPubMedGoogle Scholar
  13. 13.
    Trebing J, El-Mesery M, Schäfer V, Weisenberger D, Siegmund D, Silence K et al (2014) CD70-restricted specific activation of TRAILR1 or TRAILR2 using scFv-targeted TRAIL mutants. Cell Death Dis 5:e1035CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Bittner S, Knoll G, Füllsack S, Kurz M, Wajant H, Ehrenschwender M (2016) Soluble TL1A is sufficient for activation of death receptor 3. FEBS J 283:323–336CrossRefPubMedGoogle Scholar
  15. 15.
    Fick A, Wyzgol A, Wajant H (2012) Production, purification, and characterization of scFv TNF ligand fusion proteins. Methods Mol Biol 907:597–609CrossRefPubMedGoogle Scholar
  16. 16.
    Bossen C, Ingold K, Tardivel A, Bodmer JL, Gaide O, Hertig S et al (2006) Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human. J Biol Chem 281:13964–13971CrossRefPubMedGoogle Scholar
  17. 17.
    Chodorge M, Züger S, Stirnimann C, Briand C, Jermutus L, Grütter MG, Minter RR et al (2012) A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency. Cell Death Differ 19:1187–1195CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Division of Molecular Internal Medicine, Department of Internal Medicine IIUniversity Hospital WürzburgWürzburgGermany

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