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    Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene - methyltetrazine reactions.

    • Authors:
    • Michiro Muraki
      National Institute of Advanced Industrial Science and Technology (AIST)
      Japan
      Kiyonori Hirota
      National Institute ofAdvanced Industrial Science and Technology (AIST)
      Japan
    BMC Biotechnol 2017 07 3;17(1):56. Epub 2017 Jul 3.
    Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
    Background: Fas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem.

    Results: A procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab' domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain.

    Conclusions: The present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.
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    http://dx.doi.org/10.1186/s12896-017-0381-2DOI ListingPossible
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496246PMCFound

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