Hind Sarah Refi, Hoki Jason S, Baccile Joshua A, Boyle Patrick C, Schroeder Frank C, Martin Gregory B
Boyce Thompson Institute, Ithaca, New York.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York.
Curr Protoc Plant Biol. 2017 Sep;2:240-269. doi: 10.1002/cppb.20053. Epub 2017 Sep 1.
The field of plant receptor biology has rapidly expanded in recent years, however the demonstration of direct interaction between receptor-ligand pairs remains a challenge. Click chemistry has revolutionized small molecule research but lacks popularity in plant research. Here we describe a method that tests for the direct physical interaction of a candidate receptor protein and a peptide ligand. This protocol describes the generation of the ligand probe, transient expression of a receptor protein, enrichment of membrane-bound receptors, photo-crosslinking and click chemistry-mediated reporter addition, and detection of the receptor-ligand complex. Copper-based click chemistry confers several advantages, including the versatility to use almost any azide-containing reporter molecule for detection or visualization of the complex and addition of the reporter molecule after receptor-ligand binding which reduces the need for bulky ligand modifications that could interfere with the interaction.
近年来，植物受体生物学领域迅速发展，然而，证明受体 - 配体对之间的直接相互作用仍然是一项挑战。点击化学彻底改变了小分子研究，但在植物研究中并不流行。在这里，我们描述了一种测试候选受体蛋白与肽配体直接物理相互作用的方法。本方案描述了配体探针的生成、受体蛋白的瞬时表达、膜结合受体的富集、光交联和点击化学介导的报告分子添加，以及受体 - 配体复合物的检测。基于铜的点击化学具有几个优点，包括几乎可以使用任何含叠氮化物的报告分子来检测或可视化复合物的通用性，以及在受体 - 配体结合后添加报告分子，这减少了对可能干扰相互作用的庞大配体修饰的需求。
