title: Production of Chemokine/Chemokine Receptor Complexes for Structural Biophysical Studies.
abstract: The development of methods for expression and purification of seven-transmembrane receptors has led to an increase in structural and biophysical data and greatly improved the understanding of receptor structure and function. For chemokine receptors, this has been highlighted by the determination of crystal structures of CXCR4 and CCR5 in complex with small-molecule antagonists, followed recently by two receptor/chemokine complexes; CXCR4 in complex with vMIP-II and US28 in complex with the CX3CL1. However, these studies cover only a few of the many chemokines and chemokine receptors and production of stable receptor/chemokine complexes remains a challenging task. Here, we present a method for producing purified complexes between chemokine receptors and chemokines by coexpression in Sf9 cells. Using the complex between atypical chemokine receptor 3 and its native chemokine CXCL12 as an example, we describe the virus production, protein expression, and purification process as well as reconstitution into different membrane mimics. This method provides an efficient way of producing pure receptor/chemokine complexes and has been used to successfully produce receptor/chemokine complexes for CXC as well as CC receptors.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Gustavsson
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA. Electronic address: thandel@ucsd.edu.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Handel
mesh_terms: D000818:Animals; D001703:Biophysics / Q000379:methods*; D054377:Chemokine CXCL12 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018925:Chemokines / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D054387:Receptors, CXCR / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: C501896:ACKR3 protein, human; D054377:Chemokine CXCL12; D018925:Chemokines; D054387:Receptors, CXCR; D019707:Receptors, Chemokine; D011994:Recombinant Proteins
keywords: ACKR3; CXCL12; Chemokine; Chemokine receptor; Coexpression; Membrane protein; Protein complex; Sf9