Building a Pipeline for Producing Recombinant Affinity Reagents to Folded Proteins and Their Phosphoepitopes

ABSTRACT: While antibodies have typically served as the affinity reagent of choice for cell biologists, there has been a great deal of interest in exploiting the potential of recombinant affinity reagents. Recombinant affinity reagents have a number of practical advantages compared to polyclonal and monoclonal antibodies, such as the elimination of the need for animals, potential for genetic manipulation, the ability to be renewed at low cost, and in some instances their low molecular weights. Furthermore, they have properties that are unique, such as the ability to control affinity and specificity in vitro, the ability to subtract common epitopes, and their use as “intrabodies” inside human cells. Our goal is to devise a set of technologies that yield selective, high affinity reagents for human proteins and their post-translational modifications, in a high-throughput, low cost manner. In our experiments, we have utilized two types of scaffolds - fibronectin type III (FN3) monobodies and Forkhead Associated (FHA) domains - as affinity reagents, because it is likely that certain ones might be more successful for particular protein families and end-user applications. (FN3) monobodies are 94 amino acids in length, and have a three-dimensional structure that is convex. FHA domains are phosphothreonine-binding domains that are 100-180 residues in size, and present in prokaryotic and eukaryotic proteins. To date, we have discovered FN3 and FHA variants through phage-display that bind to a variety of natively folded, human proteins and phosphopeptide segments, respectively.