| In recent years, a new field has emerged called directed evolution, which uses the principles behind natural evolution in order to produce new functions in the laboratory from biology's most simple components: DNA, RNA, and proteins. It is an approach that relies on the fact that very rare functional sequences exist in a large pool of random or semi-random variants, and these sequences may be found through repeated cycles of selection, mutation, and replication. This thesis focuses on the development and use of mRNA display, a new in vitro method for evolving functional proteins.; Chapter 2 describes some of the contributions that have been made to the development of mRNA display during the course of this thesis. Ras was used as a model system to demonstrate that RNA-protein fusions could be made from large independently folded domains and that the protein domain was functional while fused to RNA. Also, RNA-protein fusions were found to purify efficiently with oligo(dT) cellulose, Ni-NTA, and alpha-FLAG agarose.; In order to take advantage of the improved complexity that one can achieve with mRNA display, we present an alternative method for creating combinatorial protein libraries. Chapter 3 describes a systematic process for producing highly complex libraries of proteins encoded in DNA while addressing some fundamental problems that are inherent to synthetic libraries such as insertions, deletions, and stop codons. Using this method, two randomized libraries were constructed on the basis of known protein folds.; In Chapter 4, one of these scaffolded libraries, based on the DNA-binding domain of the human retinoid-X-receptor (hRXRalpha), was mined for new function. New ATP-binding proteins were derived from the RXR-scaffolded library using mRNA display. The newly evolved proteins have micromolar affinities to ATP and show exquisite specificity among different purine analogs. The structural properties of the evolved RXR proteins are similar to the wild-type hRXRalpha, but mutational studies suggest a possible rearrangement of a zinc-finger motif.; This thesis demonstrates advances in the development and application of the mRNA display technology. The new method for designing and creating libraries can be used to produce a vast repertoire of scaffolded, random, and semi-random protein libraries. These libraries should prove important reagents not only for mRNA display but for other directed evolution techniques as well. Also, the RXR scaffold provides a new source for evolving many different types of activities, and it adds to the growing list of non-antibody scaffolds used for molecular recognition. |
