Engineering estrogen receptor-based gene switches and a superoxide dismutase for therapeutic applications

In recent years, directed evolution has rapidly emerged as a powerful approach to engineer proteins with desirable properties for application in metabolic engineering, industrial biocatalysis, and therapeutics. Despite this success, the "irrational" nature of directed evolution, where knowledge of a protein's structure-function relationship is not utilized to create mutant protein variants with altered properties, often prevents directed evolution from realizing its full potential. This dissertation describes the application of a hybrid directed evolution/rational design approach for the engineering of two different proteins with desirable properties. In the first example, stepwise saturation mutagenesis of functionally important protein sites and random point mutagenesis throughout all protein sites, accompanied by a yeast two-hybrid screening system, was used to engineer variants of the human estrogen receptor alpha (hERalpha) that bind and respond to two synthetic small molecule ligands---4,4'-dihydroxybenzil (DHB) and 2,4-di(4-hydroxyphenyl)-5ethylthiazole (L9)---with high specificity and potency. The resulting highly specific DHB/7-S receptor and L9/L7-E receptor pairs exhibit nanomolar or subnanomolar response to their respectively matched synthetic ligand, while showing negligible or no activation in response to the natural small molecule ligand for the wild type hERalpha - 17beta-estradiol. Furthermore, the two created receptor-ligand pairs do not show significant cross-interaction with each other. Both created receptor-ligand pairs have the potential to be used as gene switches for the selective regulation of gene expression in applications ranging from gene therapy and tissue engineering to metabolic engineering. In the second example, an E. coli selection system based on resistance to superoxide toxicity was used in a directed evolution approach to enhance the catalytic efficiency of a rationally designed mutant manganese superoxide dismutase enzyme exhibiting greatly reduced product inhibition and low catalytic efficiency relative to the wild type enzyme. Finally, this dissertation describes the characterization and application of a method for the creation of protein variant libraries for a rational approach to directed evolution. This method---DNA shuffling with spiked oligonucleotides (DSSO)---simultaneously randomizes multiple functionally important sites in a protein.