Purification of a heterodimeric Reelin construct to investigate binding stoichiometry
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ORIGINAL ARTICLE
Purification of a heterodimeric Reelin construct to investigate binding stoichiometry Liam S. Turk1,2,3 · Daniel Mitchell3 · Davide Comoletti1,2,3 Received: 2 July 2020 / Revised: 25 August 2020 / Accepted: 14 September 2020 © The Author(s) 2020
Abstract 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 KD 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. Keywords Protein purification · Heterodimer · Reelin · Affinity chromatography · FLAG tag · His6 tag
Introduction Reelin is a large glycoprotein that is secreted as a dimer of almost 800 kDa (D’Arcangelo et al. 1997; Kubo et al. 2002); C2101 forms an intermolecular disulfide bond in the oxidative, extracellular environment (Yasui et al. 2011). Reelin’s binding to the very-low density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2/ Special Issue: Analytical Ultracentrifugation 2019. * Liam S. Turk [email protected] * Davide Comoletti [email protected] 1
Child Health Institute of New Jersey, New Brunswick, NJ 08901, USA
2
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA
3
School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand
LRP8) is crucial to proper brain development, specifically the discrete lamination observed in the neocortex, and Reelin’s central fragment (Reelin repeats 3–6) is necessary and sufficient to induce neuronal migration during early development (Jossin et al. 2004). Not only is Reelin essential in the context of neuronal migration and layer formation in the neocortex, but it also has physiol
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