Enhanced expression, native purification, and characterization of CCR5, a principal HIV-1 coreceptor

Seven-transmembrane segment, G protein-coupled receptors (GPCRs) play impor tant roles in many biological processes in which pharmaceutical interventio n may be useful. High level expression and native purification of GPCRs are important steps in the biochemical and structural characterization of thes e molecules. Here, we describe enhanced mammalian cell expression and purif ication of a codon-optimized variant of the chemokine receptor CCR5, a GPCR that plays a central role in the entry of the human immunodeficiency virus -1 (HIV-1) into immune cells. CCR5 could be solubilized in its native state as determined by its ability to be precipitated by 2D7, a conformation-dep endent anti-CCR5 antibody, and by the HIV-1 gp120 envelope glycoprotein. Th e 2D7 antibody recognized immature and mature forms of CCR5 equally, wherea s gp120 preferentially recognized the mature form, a result that underscore s a role for posttranslational modification of CCR5 in its HIV-1 coreceptor function. The methods described herein contribute to the analysis of CCR5 and are likely to be applicable to many other GPCRs.