Semi-automated Ribosome Display for High-Throughput DARPin Binder Selection.

The combination of the Designed Ankyrin Repeat Protein (DARPin) scaffold with the selection and evolution technology of Ribosome Display has generated a wide range of specific binders to hundreds of target proteins. The binders have been used in a very diverse set of applications, from research through diagnostics to therapy. In this article, we focus on the high-throughput (HT) methods that we have developed for this purpose, which have been applied in many selections for research applications, but have never been explained in detail. We describe here the HT methods used to select for many targets in parallel, permitting the use of even different buffers and components, as well as some of the downstream characterization. To illustrate the workflow, we summarize the properties of some of the binders so obtained and their applications as well as the structures of the DARPin/target complexes. Together, these HT methods establish a robust framework for the rapid discovery and characterization of DARPins across a broad spectrum of targets.