Chu Ruiyin, Reczek David, Brondyk William
Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA.
Sci Rep. 2014 Dec 8;4:7360. doi: 10.1038/srep07360.
Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking. The resulting chip surface retained ligand binding activity and was used for monoclonal antibody kinetics assays by a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate the advantages of this whole receptor assay when compared to available peptide-based binding assays. We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utility analyzing antibodies against CD52, a GPI-anchored protein, in its native membrane environment. The results are the first demonstration of chemically stabilized chip surfaces for membrane protein SPR assays.
通过表面等离子体共振测量抗体与完整膜蛋白的结合动力学一直具有挑战性，这主要是因为在芯片表面捕获膜蛋白并保持其天然构象存在固有的困难。在此，我们描述了一种方法，其中通过亲和标签纯化带有His标签的GPCR（CXCR5）并将其捕获在Biacore芯片表面。然后通过有限交联将捕获的受体蛋白稳定在芯片表面。所得芯片表面保留了配体结合活性，并通过标准的Biacore动力学测定方法和简单的低pH再生步骤用于单克隆抗体动力学测定。与现有的基于肽的结合测定相比，我们证明了这种全受体测定的优势。我们进一步将捕获 - 稳定方法的应用扩展到病毒样颗粒，并证明了其在天然膜环境中分析针对GPI锚定蛋白CD52的抗体的效用。这些结果首次证明了用于膜蛋白SPR测定的化学稳定芯片表面。
