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

BMC Biotechnology

Research Article

Kiyonori Hirota¹  Michiro Muraki¹ 

[1] Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, 305-8566, Tsukuba, Ibaraki, Japan;

关键词: Human Fas ligand;    Extracellular domain;    Site-specific conjugation;    trans;    Methyltetrazine;    Fluorochrome;    Functional protein;    Receptor-binding activity;   

DOI  :  10.1186/s12896-017-0381-2

 received in 2017-04-05, accepted in 2017-06-27,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundFas 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.ResultsA 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.ConclusionsThe 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.

【 授权许可】

CC BY   
© The Author(s). 2017