Lang Isabell, Kums Juliane, Wajant Harald
Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070, Würzburg, Germany.
Methods Mol Biol. 2017;1557:63-77. doi: 10.1007/978-1-4939-6780-3_7.
The quantitative evaluation of the interaction of soluble CD95L with CD95 is not only important for a detailed understanding of CD95 biology but is also of special relevance for the characterization and development of inhibitors of this interaction. The assembly of a CD95L-CD95 complex capable to recruit intracellular factors not only involves pre-assembly of CD95 molecules in the absence of CD95L but is also modulated by cellular factors such as interaction with the actin cytoskeleton and plasma membrane compartmentation of CD95. Due to these influential variables cell-free methods allow only an inadequate analysis of CD95L binding to cell expressed CD95. To enable easy, sensitive and highly reproducible cellular binding studies for the investigation of the CD95L-CD95 interaction, we generated fusion proteins of soluble CD95L with the luciferase from Gaussia princeps (GpL). The GpL domain contained in the GpL-CD95L fusion proteins does not interfere with CD95 binding and makes the GpL-CD95L fusion proteins highly suitable for cellular binding studies and tracer applications. In this chapter, we report detailed protocols for the production of GpL-CD95L fusion proteins and their use in cellular binding studies.
可溶性CD95L与CD95相互作用的定量评估不仅对于详细了解CD95生物学特性很重要，而且对于表征和开发这种相互作用的抑制剂也具有特殊意义。能够募集细胞内因子的CD95L-CD95复合物的组装不仅涉及在没有CD95L的情况下CD95分子的预组装，还受到细胞因子的调节，如与肌动蛋白细胞骨架的相互作用以及CD95的质膜分隔。由于这些影响变量，无细胞方法仅能对CD95L与细胞表达的CD95的结合进行不充分的分析。为了能够进行简单、灵敏且高度可重复的细胞结合研究以探究CD95L-CD95相互作用，我们构建了可溶性CD95L与高斯海萤荧光素酶（GpL）的融合蛋白。GpL-CD95L融合蛋白中所含的GpL结构域不干扰CD95结合，使得GpL-CD95L融合蛋白非常适合用于细胞结合研究和示踪应用。在本章中，我们报告了GpL-CD95L融合蛋白的详细制备方案及其在细胞结合研究中的应用。
