Protein engineering by Bordetella bronchiseptica bacteriophage

Recognition scaffolds for discovering ligands for targets rely on molecular display technologies to identify high affinity, specific binding partners. Combinatorial protein engineering to discover proteins with novel binding specificities and desired properties has developed into a powerful technology. Mimicking the immune system, the potent combination of diverse molecular recognition scaffolds and display on evolvable platforms provides a rapid approach to the discovery of ligands and receptors for new targets. The research described herein aims to extend the use of phage-displayed peptides and proteins for engineering novel binding scaffolds for biotechnological and biomedical applications. Chapter One reviews promising non-antibody protein scaffolds as a starting point for the design of new binding proteins. The ideal protein generating machine, which can itself generate and display diverse repertoires of proteins, has been found in the genome of the Bordetella bronchiseptica bacteriophage (BP). Chapter Two reports progress toward harnessing BP for development of self-made phage libraries (SMPL), which can display trillions of peptides on the phage tail fibers. BP generates diversity using a diversity generating retroelement (DGR). Chapter Three reviews the three proteins essential for the function of DGR. This chapter lays the groundwork for further characterization of the three proteins based on expression experiments. Lastly, Chapter Four combines the proven success of structured libraries to identify binders for specific targets using a mega random peptide library (MRPL). The MRPL is the most diverse phage-displayed peptide library reported and has generated successful binders to numerous and diverse targets. Thus, this dissertation provides a link between engineered M13 filamentous phage display technologies and the natural diversity generating capabilities of BP.