mRNA display for the in vitro evolution ofartificial proteins and enzymes

Artificial proteins and enzymes have the potential to aid in the production of pharmaceuticals and to facilitate basic biomedical research. Two methods currently exist for the development of artificial proteins: rational design and de novo selection. Rational design requires detailed knowledge of enzyme catalysis in order to design an enzyme active site in silico, and then introduce this active site into a protein. However, gaps in the understanding of protein folding and structure-function relationships make this approach challenging and far from routine. In contrast, laboratory evolution approaches to isolate artificial proteins and enzymes from libraries of variants are well established. In vitro selection techniques are powerful tools for the exploration of large areas of sequence space (up to 1013 unique sequences) in the search for functional proteins and enzymes. mRNA display selection methods have only recently been developed, and the application of this technique for the engineering of de novo enzymes has not been fully explored. This thesis describes the establishment of an mRNA display platform for the selection and evolution of novel proteins and enzymes from large, high-diversity libraries. The synthesis of novel selection substrates are described that will facilitate the application of mRNA display to the selection of Diels-Alderase enzymes. A novel application of mRNA display is described for the solution-phase selection of protein-ligand pairs using interaction-dependent reverse transcription. Further development of this research could increase the throughput of ligand discovery to complement the pace at which new macromolecular targets of interest are being discovered. The ability to generate tailor-made enzymes that catalyse novel reactions is of considerable interest. The applications of mRNA display selection described in this thesis will help to extend the range of enzyme catalysis, and to elucidate basic mechanisms of biocatalysis and protein evolution. Moreover, such ‘designer enzymes’ hold promise for a huge range of applications including, but not limited to, the synthesis of chemicals, pharmaceuticals, and production of renewable fuels.