C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue PLoS ONE Année : 2014

C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.

Résumé

Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+) channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

Dates et versions

hal-01073781 , version 1 (10-10-2014)

Identifiants

Citer

Antoine Picciocchi, Lina Siaučiūnaiteė-Gaubard, Isabelle Petit-Hartlein, Rabia Sadir, Jean Revilloud, et al.. C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.. PLoS ONE, 2014, 9 (1), pp.e87394. ⟨10.1371/journal.pone.0087394⟩. ⟨hal-01073781⟩
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