Two Dimensional Screening : Towards Establishing a Novel Technique to Study Biomolecular Interactions

Abstract

In all combinatorial approaches used so far one (macro-) molecule of a given structure is used to select the best binding partner from a library of potential ligands or acceptors. Therefore, only a certain, limited, part of the structure activity space is evaluated. By combining a library of molecules derived from one particular acceptor structure with a library of potential ligands, a significantly larger part of the structure activity space of this particular pair of molecules is accessed. The insight gained in the interaction of interest in this way is expected to be more detailed. This thesis aimed at establishing practical ways to combine a library of peptide ligands and a library of protein acceptors in the context of one particular model system. A library of cellulose bound peptides and a library of bacteriophage displayed variants of one protein were combined. Calmodulin (CaM) was chosen as a model protein. In binding tests aiming at the design of a peptide ligand library remarkable short CaM binding peptides were found. Furthermore, it was possible to present biological active CaM on the surface of M13-bacteriophage. A highly mutated CaM-library was created using the error prone PCR and displayed on the surface of bacteriophage. Using this CaM-library in affinity enrichment experiments with peptides and peptide libraries new variants of CaM were enriched. The results presented in this thesis show that two libraries can be combined. Furthermore it was demonstrated that Two Dimensional Screening is with the chosen approach not only feasible but resulted in novel results, which would be difficult to reach with a different approach.