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首页> 外文期刊>BMC Biotechnology >Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene – methyltetrazine reactions
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Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene – methyltetrazine reactions

机译：使用反式-环辛烯-甲基四嗪反应的人类Fas配体胞外域的位点特异性化学缀合

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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.
机译：背景Fas配体作为主要的细胞死亡诱导蛋白，在人类免疫系统中起着关键作用。人类Fas配体（hFasLECD）的胞外域触发了恶性细胞的凋亡，因此有望在医学生物技术中具有巨大潜力。但是，由于缺乏相对于包括全身给药的副作用在内的益处，因此该蛋白质目前在临床医学中的应用受到阻碍。需要通过附加有用的附加功能对蛋白质进行工程改造的有效程序，以解决该问题。结果利用反式环辛烯基和甲基四嗪基团的电子反需求Diels-Alder反应，设计了hFasLECD与荧光染料和功能蛋白进行位点特异性化学偶联的程序。与先前研究中使用马来酰亚胺衍生物进行的常规化学修饰反应相比，本研究中的共轭反应是通过使用摩尔过量的待连接化合物来实现的。分离的hFasLECD与磺基-Cy3，抗生物素蛋白和兔IgG Fab'结构域的结合物表现出附着分子的功能和结构完整性，而不会损害对人Fas受体胞外结构域的特异性结合活性。结论本研究为生产具有额外有益功能的工程化hFasLECDs提供了新的基本策略，通过使用这种蛋白作为新型分子工具的一部分，将导致改进的诊断系统和有效治疗严重疾病的方法的发展。

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