Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

We previously designed a new family of artificial proteins named aRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized aRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a ‘‘filtration’ ’ procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.76109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific aRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between aRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that aRep library is a new and versatile source of tight and specific binding proteins wit