Capture-stabilize approach for membrane protein SPR assays

Chu, Ruiyin; Reczek, David; Brondyk, William

With this ''capture-stabilize'' approach, purified GPCR molecules are first captured directly on the Ni-NTA chip surface via a C-terminal 6xHis-tag. Alternatively, an anti-6xHis antibody can be first immobilized on a CM5 chip, and then similar levels of the receptor protein are captured indirectly on the chip surface. If necessary, more receptor can be captured to meet specific requirements, such as small molecule compound binding assays. In both cases, receptor molecules are captured specifically via the 6xHis-tag. The subsequent limited cross-linking step further stabilizes the captured receptor molecules. Therefore, it is essential that the starting material, purified GPCR protein, is tested for activity by ligand binding. The purification methods used here, adapted from Jaakola V. et al. for determin-ing the crystal structure of the human A2A adenosine receptor in complex with a high-affinity subtype-selective antagonist 16 , yield active pure GPCRs without the need to engineer stabilized receptors which require a substantial amount of effort 15 . During the stabilization step, some of the receptor binding sites may have been blocked by the cross-linking reaction. A 10-fold dilution of the standard amine-coupling reagent from GE (200 mM NHS/50 mM EDC) gave the best results. Over cross-linking with 200 mM NHS/50 mM EDC resulted in significant loss of binding activity (data not shown). We also tested five other cross-linking reagents for receptor stabilization on both NTA and CM5 chip surfaces. In each case, similar results were obtained (data not shown). Therefore, we chose the 20 mM NHS/5 mM EDC as our standard stabilization reagent. The receptor surface stabilized by this simple cross-linking step yielded sufficient binding capacity for analyzing both antibody and ligand binding. More importantly, the limited cross-linking step kept the hCXCR5 whole receptor surface stable. The surface was not only resistant to the surfactant (0.05% Tween-20) present in the running buffer, but also allows for low pH (pH 1.5) regeneration steps. These features enabled the binding assay to be performed with the same conditions used for soluble proteins, which resulted in high quality kinetics sensorgrams and excellent reproducibility (CV,25%). This assay has been applied successfully to eight different GPCR targets (data not shown) and is expected to be applicable to most, if not all, GPCR molecules.