Enhanced expression, native purification, and characterization of CCR5, a principal HIV-1 coreceptor.
Seven-transmembrane segment, G protein-coupled receptors (GPCRs) play important roles in many biological processes in which pharmaceutical intervention may be useful. High level expression and native purification of GPCRs are important steps in the biochemical and structural characterization of these molecules. Here, we describe enhanced mammalian cell expression and purification 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-dependent anti-CCR5 antibody, and by the HIV-1 gp120 envelope glycoprotein. The 2D7 antibody recognized immature and mature forms of CCR5 equally, whereas gp120 preferentially recognized the mature form, a result that underscores 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.
Mirzabekov
T
T
Department of Cancer Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Bannert
N
N
Farzan
M
M
Hofmann
W
W
Kolchinsky
P
P
Wu
L
L
Wyatt
R
R
Sodroski
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D019713:Receptors, CCR5 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D015699:HIV Envelope Protein gp120; D019713:Receptors, CCR5