title: Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation.
abstract: BACKGROUND
  Human Fas ligand extracellular domain has been investigated as an important target protein in the field of medical biotechnology. In a recent study, the author developed an effective method to produce biologically active human Fas ligand extracellular domain derivatives using site-specific chemical modifications.
  
  
  RESULTS
  A human Fas ligand extracellular domain derivative containing a reactive cysteine residue within its N-terminal tag sequence, which locates not proximal to the binding interface between the ligand and the receptor in terms of the three-dimensional structure, was modified by Fluorescein-5-Maleimide without impairing the specific binding activity toward human Fas receptor extracellular domain. The purified protein sample free of low molecular-weight contaminants showed a characteristic fluorescence spectrum derived from the attached Fluorescein moieties, and formed a stable binding complex with human Fas receptor extracellular domain-human IgG1 Fc domain fusion protein in solution. The conjugation number of the fluorochrome was estimated to be 2.5 per a single human Fas ligand extracellular domain trimer from the ratio of the absorbance value at 280 nm to that at 495 nm.
  
  
  CONCLUSIONS
  A functional fluorescent human Fas ligand extracellular domain derivative was prepared via a site-specific conjugation of fluorochrome, which was guided by the three-dimensional structure information on the ligand-receptor complex. Fluorescent derivatives created by this method may contribute to the development of an improved diagnosis system for the diseases related to Fas receptor.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, 305-8566 Japan.
authors.forename: Michiro
authors.identifier: 0000-0003-4953-8007
authors.initials: M
authors.lastname: Muraki
keywords: Extracellular domain; Fluorochrome; Human Fas ligand; Receptor-binding activity; Site-specific conjugation; Three-dimensional structure