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Purification of a heterodimeric Reelin construct to investigate binding stoichiometry

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journal contribution
posted on 04.08.2022, 22:19 authored by LS Turk, D Mitchell, Davide ComolettiDavide Comoletti
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.

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/s00249-020-01465-6

Journal title

European Biophysics Journal

Volume

49

Issue

8

Publication date

01/12/2020

Pagination

773-779

Publisher

Springer Science and Business Media LLC

Publication status

Published

Online publication date

14/10/2020

ISSN

0175-7571

eISSN

1432-1017

Language

en