Preferences help
enabled [disable] Abstract
Number of results
2008 | 55 | 1 | 51-56
Article title

Cloning and purification of functionally active Fas ligand interfering protein (FIP) expressed in Escherichia coli

Title variants
Languages of publication
This report presents purification and characterization of the extracellular domain of rat Fas protein, called FIP (FasL interfering protein), expressed as inclusion bodies in Escherichia coli. FIP was extracted from the inclusion bodies, solubilized with 8 M urea, purified by a single-step immobilized metal ion (Ni2+) affinity chromatography and refolded. SDS/PAGE and mass spectrometry analysis of the purified protein verified its purity. Fluorescence spectrum analysis showed that the refolding procedure caused structural changes which presumably might have led to oligomerization. The purified FIP has biological activities: it binds specifically soluble Fas ligand and protects human Jurkat lymphocytes against FasL-dependent apoptosis. This efficient procedure of FIP expression in E. coli and renaturation may be useful for production of therapeutically important proteins.
Physical description
  • Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warszawa, Poland
  • Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warszawa, Poland
  • Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warszawa, Poland
  • Barnhart BC, Legembre P, Pietras E, Bubici E, Franzoso G, Peter ME (2004) CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells. EMBO J 23: 3175-3185.
  • Blum H, Bayer H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93-99.
  • Cheng J, Zhou T, Liu C, Shapiro JP, Brauer MJ, Kiefer MC, Barr PJ, Mountz DJ (1994) Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 263: 1759-1762.
  • Choi C, Benveniste EN (2004) Fas ligand/Fas system in the brain: regulator of immune and apoptotic responses. Brain Res Brain Res Rev 44: 65-81.
  • Ciesielski-Treska J, Ulrich G, Taupenot L, Chasserot-Golaz S, Zwiller J, Revel MO, Aunis D, Bader MF (2001) Mechanisms underlying neuronal death induced by chromogranin A-activated microglia. J Biol Chem 276: 13113-13120.
  • Curtin JF, Cotter TG (2003) Live and let die: regulatory mechanisms in Fas-mediated apoptosis. Cell Signal 15: 983-992.
  • Ethell DW, Buhler LA (2003) Fas ligand-mediated apoptosis in degenerative disorders of the brain. J Clin Immunol 23: 363-370.
  • Green DR (2003) Introduction: apoptosis in the development and function of the immune system. Semin Immunol 15: 121-123.
  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714-2723.
  • Itoh N, Nagata S (1993) A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J Biol Chem 268: 10932-10937.
  • Krammer PH (2000) CD95's deadly mission in the immune system. Nature 407: 789-795.
  • Li Y, Yang X, Nguyen AH, Brockhausen I (2007) Requirement of N-glycosylation for the secretion of recombinant extracellular domain of human Fas in HeLa cells. Int J Biochem Cell Biol 39: 1625-1636.
  • Nagata S (1997) Apoptosis by death factor. Cell 88: 355-365.
  • Peitsch MC (1995) Protein modeling by E-mail. Bio/Technology 13: 658-660.
  • Raoul C, Henderson CE, Pettmann B (1999) Programmed cell death of embryonic motoneurons triggered through the Fas death receptor. J Cell Biol 147: 1049-1061.
  • Ryan AE, Shanahan F, O`Connell J, Houston AM (2005) Addressing the 'Fas counterattack' controversy: blocking fas ligand expression suppresses tumor immune evasion of colon cancer in vivo. Cancer Res 65: 9817-9823.
  • Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME (1998) Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17: 1675-1687.
  • Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31: 3381-3385.
  • Smith CA, Farrah T, Goodwin RG (1994) The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76: 959-962.
  • Starling GC, Bajorath J, Emswiler J, Ledbetter JA, Aruffo A, Kiener PA (1997) Identification of amino acid residues important for ligand binding to Fas. J Exp Med 185: 1487-1492.
  • Starling GC, Kiener PA, Aruffo A, Bajorath J (1998) Analysis of the ligand binding site in Fas (CD95) by site-directed mutagenesis and comparison with TNFR and CD40. Biochemistry 37: 3723-3726.
  • Wajant H, Pfizenmaier K, Scheurich P (2003) Non-apoptotic Fas signaling. Cytokine Growth Factor Rev 14: 53-66.
  • Zawadzka M, Kaminska B (2005) A novel mechanism of FK506-mediated neuroprotection: downregulation of cytokine expression in glial cells. Glia 49: 36-51.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.