首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Large-scale expression and purification of the major HIV-1 coreceptor CCR5 and characterization of its interaction with RANTES
Authors:Nisius Lydia  Rogowski Marco  Vangelista Luca  Grzesiek Stephan
Institution:aDivision of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland;bUnit of Human Virology, DIBIT, San Raffaele Scientific Institute, Via Olgettina 58, Milan, Italy
Abstract:The G protein-coupled receptor CCR5 is a human chemokine receptor involved in the activation and migration of leukocytes. CCR5 is also the major HIV-1 coreceptor that, together with human CD4 and the viral glycoprotein gp120, promotes virus entry into host cells. Thus inhibition of the CCR5-gp120 interaction presents a promising route to prevent HIV infections. Atomic structural details of the interaction between CCR5 and its cognate chemokines or gp120 are presently unknown due to the general difficulties of membrane protein structure determination. Here, we report the high-yield expression of human CCR5 in baculovirus-infected Sf9 insect cells. Highly purified (>90%) CCR5 is obtained in detergent-solubilized form at yields of about 1 mg/l cell culture. The conformational integrity of recombinant CCR5 after purification is shown by immunoprecipitation with the conformation-dependent monoclonal antibody 2D7, CD and NMR spectroscopy. The detergent micelles contain CCR5 in monomeric and dimeric forms, which can be separated by size exclusion chromatography and characterized individually. Further functional characterization by isothermal titration calorimetry indicates that the recombinant receptor interacts with its cognate chemokine RANTES. This interaction is strongly suppressed when sulfation of CCR5 is inhibited in the insect cells.
Keywords:CCR5  HIV-1  GPCR  Membrane protein  RANTES  Secondary structure  NMR  ITC  CD  Baculovirus  Sf9 cells
本文献已被 ScienceDirect PubMed 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号