Date of Award

6-15-2026

Document Type

Thesis

Publisher

Santa Clara : Santa Clara University, 2026

Department

Bioengineering

First Advisor

Zhiwen Zhang

Abstract

The principal objective of this paper is to provide a proof-of-concept of a novel protein engineering method called Molecular Devolution (MD). MD is an evolutionary protein engineering method that reduces the size of enzymes while retaining its functional properties. This paper outlines an MD protocol for Cre Recombinase. Cre Recombinase is an enzyme that is part of the Cre LoxP DNA Recombination System, which is a leading gene editing system widely used in genetic research. This paper outlines a MD approach that was used to miniaturize Cre Recombinase and remove unnecessary complexities, allowing for simpler and more e!cient use in research settings as well as therapeutic applications in the future. MD involves structural analysis to identify the active site of the enzyme, DNA library construction, genetic transformation and functional screening. The Cre Recombinase active site was identified to include several key residues. A DNA library, preserving the active site, while adding randomized flanking sequences was synthesized. The DNA library along with a reporter gene was transformed into E. Coli. Screening was conducted with a Red Fluorescent Protein (RFP) assay, and fluorescence was observed in several transformed E. Coli colonies. Two variants showed particularly strong fluorescence, and were sequenced and analyzed in AlphaFold and Pymol. The structural analysis results showed retention of the active site while a significant reduction in non-essential residues, overall support the e!cacy of MD as a protein miniaturization tool.

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