期刊
ANALYTICAL BIOCHEMISTRY
卷 339, 期 2, 页码 271-281出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2004.12.017
关键词
biacore; surface plasmon resonance; GPCR; membrane receptor; HIV
资金
- NIAID NIH HHS [P30 AI28691] Funding Source: Medline
- NIGMS NIH HHS [P01 GM66521] Funding Source: Medline
Establishing solubilization conditions for membrane-associated receptors is often a tedious empirical process. Here we describe a novel application of SPR biosensor technology to screen solubilization conditions automatically and to assess receptor activity directly. We focus On two chemokine receptors, CXCR4 and CCR5, which are important in HIV cell invasion. The autosampler in Biacore 3000 permitted whole cells expressing C-terminally tagged receptors to be automatically lysed Under a given solubilization condition and the Mates to be injected over an antibody surface. The total amount of solubilized receptor could be quantitated from the antibody capture level, whereas the amount of active receptor could be quantitated Using a subsequent injection of conformationally sensitive antibody or protein. Using this approach, we identified detergent/lipid/buffer combinations that enhanced and maintained receptor activity. We also used the biosensor to demonstrate CD4-dependent binding of gp120 to solubilized CCR5 and to develop affinity chromatography-based purification methods that increased receptor activity more than 300%. Together, these results illustrate the benefits Of using, the biosensor as a tool for isolating functional membrane receptors and for analyzing ligand receptor interactions. (c) 2004 Elsevier Inc. All rights reserved.
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