Hengelbrok_Helena.pdf

Abstract

Natural enzymes are inherently limited by evolutionary history. Shared protein scaffolds, active site residues, and functional domains across large protein superfamilies demonstrate the kinship, and therefore the limitations, of natural enzymes. In order to explore outside of nature’s boundaries, completely non-natural proteins must be created and investigated. Previous work in the Hecht lab has led to the creation of SynF4, a de novo enzyme that has no homology to any natural protein. SynF4 will be used as the basis for the design, construction, and characterization of a library of de novo proteins. As the parental sequence is an enzyme, it is proposed that some library members will themselves have enzymatic activity. To introduce diversity, a combinatorial approach was used to selectively and specifically replace first and second shell residues of the SynF4 active site. Next generation sequencing revealed the library was successfully assembled and resulted in 4.7 x 10^6 unique sequences that closely mimic the design. Initial characterization of the library demonstrated that the library is well expressed and soluble in E. coli. Purification of two randomly chosen library members 4H and 6F was facile. Both proteins were shown to be dimeric, well folded, and stable as designed; this suggest that other library members will share these desired characteristics. Overall, the assembled library is highly diverse and closely matches the design. Probing for functional enzymes was initiated, and a refined methodology is presented. With further investigation, functional de novo enzymes might be discovered within the assembled library. The discovery of more de novo enzymes will provide insight into previously unexplored sequence space and enzymatic reaction mechanisms.