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

Chemotactic factor that attracts monocytes

Chemokine 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 its 15N-,
15N/13C-, 2H/15N/13C- isotope-labeled derivatives, at milligram levels using E.
coli expression system. Purified CCL7 not only maintains a well-folded
three-dimensional structure as analyzed using circular dichroism and 1H/15N NMR
but it also induces coupling of heterotrimeric G-proteins and b-arrestins for
selected chemokine receptors in cellular system. We compared cAMP response
induced by histidine tagged CCL7 and native CCL7 and found that modification of
the N-terminus of CCL7 compromises its functionality. 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.

Department of structural biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, 208016, India

This entry was used to transfer NMR assignment

303 oK
pH 5.1 (KPi 6.12 titrated by d4-AcOD)
20mM KPi
0.4mM MES
7 mM NaCl