20 Turk et al European_Biophysics_Journal.pdf (1.11 MB)
Download filePurification of a heterodimeric Reelin construct to investigate binding stoichiometry
journal contribution
posted on 2022-08-04, 22:19 authored by LS Turk, D Mitchell, Davide ComolettiDavide ComolettiReelin 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.
Funding
Collaborative Research: Structure and Function of Reelin in Brain Development | Funder: RUTGERS UNIVERSITY | Grant ID: 1755189
History
Preferred citation
Turk, L. S., Mitchell, D. & Comoletti, D. (2020). Purification of a heterodimeric Reelin construct to investigate binding stoichiometry. European Biophysics Journal, 49(8), 773-779. https://doi.org/10.1007/s00249-020-01465-6Publisher DOI
Journal title
European Biophysics JournalVolume
49Issue
8Publication date
2020-12-01Pagination
773-779Publisher
Springer Science and Business Media LLCPublication status
PublishedOnline publication date
2020-10-14ISSN
0175-7571eISSN
1432-1017Language
enUsage metrics
Categories
Keywords
Protein purificationHeterodimerReelinAffinity chromatographyFLAG tagHis6 tagCell Adhesion Molecules, NeuronalExtracellular Matrix ProteinsHEK293 CellsHumansNerve Tissue ProteinsNeuronsProtein BindingProtein MultimerizationProtein Structure, QuaternaryReelin ProteinSerine EndopeptidasesSignal Transduction1 Underpinning research1.1 Normal biological development and functioningBiophysicsPhysical Sciences not elsewhere classifiedAtomic, Molecular, Nuclear, Particle and Plasma Physics not elsewhere classifiedBiochemistry and Cell Biology not elsewhere classified