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

Abstract

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep 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 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep 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 αRep 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 αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools

“…We thus selected two kinds of tailor-made binders boasting excellent track records: nanobodies (Domanska et al, 2011;Korotkov et al, 2009;Rasmussen et al, 2011) (also called VHH antibody fragments) and aRep (Ferrandez et al, 2014;Guellouz et al, 2013;Urvoas et al, 2010) proteins. The former, which are well known, are single-domain antibody fragments derived from a Camelidae species; the latter constitute a relatively new family of artificial protein binders based on natural HEAT repeat proteins (Guellouz et al, 2013;Urvoas et al, 2010).…”

“…The former, which are well known, are single-domain antibody fragments derived from a Camelidae species; the latter constitute a relatively new family of artificial protein binders based on natural HEAT repeat proteins (Guellouz et al, 2013;Urvoas et al, 2010). We obtained seven monoclonal nanobodies (Nb) after a llama immunization with 1 mg of Octarellin V.1 (see Section 4) and were able to isolate stable complexes with all of them, using size exclusion chromatography.…”

“…The selection was done as previously described using aRep library 2.1 (Guellouz et al, 2013;Tiouajni et al, 2014) using 40 mg/mL of Octarellin V.1 coated on a micro-titer ELISA plate. Positive aRep genes were sub-cloned in the pQE81L vector (Qiagen) for aRep production and purification.…”

Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets

“…(Guellouz et al 2013;Urvoas et al 2010). Phages from each library were prepared using XL1-Blue MRF' bacteria transformed with the phagemid libraries and infected with the helper phage Phaberge (Soltes et al 2003).…”

“…aReps are efficiently expressed and folded, and very stable. Large libraries have been recently described (Guellouz et al 2013), which have allowed the selection of tight and specific binders against a range of different and unrelated soluble proteins, with dissociation constants, K D , in the nanomolar to micromolar range. Crystallographic structures of aRep/target protein complexes show conformational-specific recognition (Guellouz et al 2013).…”