Reelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein’s function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin’s central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin’s known receptors, calculating the K_D of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.

Reelin是一种分泌的糖蛋白，在新皮层发育和突触功能中不可或缺。Reelin以同型二聚体的形式存在，其两条链在半胱氨酸2101处通过二硫键连接，该特征对蛋白质的功能至关重要。这点突出了一个事实，即即使Reelin的突变（C2101A）单体构建体保留了与其受体结合的能力，只有二聚体Reelin才能引发有效的规范信号转导。受体簇已被证明在信号通路中很重要，但是缺乏关于Reelin-受体结合相互作用的化学计量的直接证据。在这里，我们描述了异二聚体Reelin构建体的构建和纯化，以研究Reelin-受体结合的化学计量及其如何影响Reelin途径信号传导。我们已设计出不同的策略并完成了使用差异标记和串联亲和色谱法生产Reelin中心片段异二聚体的方案，以使链A在氨基酸序列中为野生型，而链B包含受体结合位点突变（K2467A）。我们还验证了异二聚体能够与Reelin已知受体之一的胞外域结合，从而计算K_D的互动。这种异二聚体构建体将使我们能够更详细地了解Reelin与它的已知受体相互作用并启动途径信号传导的机制。