Large-scale expression and purification of the major HIV-1 coreceptor CCR5 and characterization of its interaction with RANTES

Lydia Nisius, Marco Rogowski, Luca Vangelista, Stephan Grzesiek

Research output: Contribution to journalArticle

26 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)155-162
Number of pages8
JournalProtein Expression and Purification
Volume61
Issue number2
DOIs
Publication statusPublished - Oct 1 2008

Keywords

  • Baculovirus
  • CCR5
  • CD
  • GPCR
  • HIV-1
  • ITC
  • Membrane protein
  • NMR
  • RANTES
  • Secondary structure
  • Sf9 cells

ASJC Scopus subject areas

  • Biotechnology

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