Solubilization, stabilization, and purification of chemokine receptors using biosensor technology.
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 lysates 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.
Navratilova
Iva
I
Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA.
Sodroski
Joseph
J
Myszka
David G
DG
D001331:Automation; D015374:Biosensing Techniques / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods; D004355:Drug Stability; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D011487:Protein Conformation; D019713:Receptors, CCR5 / Q000302:isolation & purification / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000302:isolation & purification; D019707:Receptors, Chemokine / Q000302:isolation & purification*; D012995:Solubility; D020349:Surface Plasmon Resonance / Q000379:methods*
D015699:HIV Envelope Protein gp120; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D019707:Receptors, Chemokine