Purification of native CCL7 and its functional interaction with selected chemokine receptors

Abstract

AbstractChemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its15N-,15N/13C-,2H/15N/13C-isotope-labeled derivatives, at milligram levels usingE. coliexpression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and β-arrestins for selected chemokine receptors in cellular system. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies.