Capture-stabilize approach for membrane protein SPR assays

Chu, Ruiyin; Reczek, David; Brondyk, William

Using unmodified receptor coding sequence, we added a 6xHis [18] [19] [20] and HPC4 21, 22 tandem tag to the C-terminus of the receptor to facilitate both purification and Biacore chip capturing processes. The tagged human CXCR5 receptor protein was expressed in insect Sf9 cells using the baculovirus expression system. The recombinant receptor protein was purified by a 2-step Ni-NTA/HPC4 affinity purification procedure, and then captured on a Biacore NTA chip surface 18, 19 . In order to regenerate the receptor surface for antibody binding kinetics assay, a limited cross-linking 23 step was applied to stabilize the captured receptor. The resulting receptor surface retained ligand binding activity and was used for monoclonal antibody kinetics analysis by employing a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate here the advantages of this capture-stabilize approach in enabling a whole receptor-based assay. We further extended the application of the capture-stabilize approach to virus-like particles 24, 25 and demonstrate its utilities analyzing antibodies against CD52, a GPIanchored protein, in its native membrane environment. Results Peptide-based binding kinetics assay. If an antibody binds to a linear portion of a GPCR molecule, for example, a contiguous sequence in the N-terminal extracellular domain, a synthetic peptide can be used for rapid binding evaluation. Two different assay formats can be used: the conventional format in which peptide flows over captured antibody at different concentrations or the ''reverse format'' which flows antibody over the immobilized peptide. Three anti-hCXCR5 mAb clones, 16D7, 79E7 and MAB190, were used to evaluate and compare these assay formats. As shown in Figure 1 , in the conventional format, reproducible kinetics data (Table 1) were obtained. However, it did not provide sufficient resolution and sensitivity to differentiate the 3 mAb clones which is reflected in the Kon-Koff rate map shown in Figure 1a . In contrast, the reversed peptide-based assay format was able to distinguish mAb 16D7 with an almost 10-fold slower Koff-rate (Table 1) , which was not revealed by the conventional format assay. The Kon-Koff rate map (Figure 1b ) also indicated that this reversed peptide-based assay format displayed good reproducibility.