Isolation and Characterization of "Orphan-RTK" Ligands Using an Integrated Biosensor Approach

摘要

AbstractThe interest in biosensor-based ligand isolation strategies is to a large extent, a result of fundamental changes in the way novel proteins are isolated and characterized. Advances in genetic screening techniques have led to the identification of novel protein families with little knowledge of function or physiological context (1,2) and a rapidly increasing number of these so-called "orphan proteins" calls for complementary strategies to elucidate their structure and function. Over the past few years, different approaches have been developed to isolate ligands solely on the basis of their affinity for a particular orphan receptor. These include expression cloning strategies to detect cell-membrane bound ligands with tagged receptor extracellular domain (ECD) fusion proteins (3–6) and cell rescue assays to detect soluble ligands (7–9). BIAcore technology has been applied to search for suitable ligand sources of orphan receptors (10–12), but is also well suited to integrated use within a "classical" purification scheme (13,14) and has been exploited as an affinity detector to facilitate the isolation of orphan proteins (15,16). The advantages of this technology include a fast, robot-driven assay that is unaffected by sample toxicity, and the ability to assess specificity and kinetics of the observed interaction even in relatively crude samples, thus minimizing the risk of false positives.KeywordsBinding BufferElution BufferOrphan ReceptorAffinity SurfaceCoupling BufferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.