Production of Chemokine/Chemokine Receptor Complexes for Structural Biophysical Studies
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Production of Chemokine/Chemokine Receptor Complexes for Structural Biophysical Studies. / Gustavsson, Martin; Zheng, Yi; Handel, Tracy M.
I: Methods in Enzymology, Bind 570, 2016, s. 233-60.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Production of Chemokine/Chemokine Receptor Complexes for Structural Biophysical Studies
AU - Gustavsson, Martin
AU - Zheng, Yi
AU - Handel, Tracy M
N1 - © 2016 Elsevier Inc. All rights reserved.
PY - 2016
Y1 - 2016
N2 - 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.
AB - 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.
KW - Animals
KW - Biophysics/methods
KW - Chemokine CXCL12/chemistry
KW - Chemokines/genetics
KW - Protein Engineering/methods
KW - Receptors, CXCR/chemistry
KW - Receptors, Chemokine/genetics
KW - Recombinant Proteins/genetics
KW - Sf9 Cells
U2 - 10.1016/bs.mie.2015.10.003
DO - 10.1016/bs.mie.2015.10.003
M3 - Journal article
C2 - 26921949
VL - 570
SP - 233
EP - 260
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
ER -
ID: 329434288