Directed Evolution of Haloalkane Dehalogenase Enzymes using Ancestral Protein Sequence Reconstruction

Voiceover

Presenter(s)(s)

Ashley Maloney

Abstract or Description

Ancestral sequence reconstruction (ASR) is a computational technique that engineers thermodynamically stable and robust proteins for numerous industrial bioengineering applications such as, pharmaceuticals and medicine. ASR constructs a phylogenetic tree that leverages multiple sequence alignment data to illustrate possible ancestral protein sequences based on the modern collection of amino acid sequences. With a wide range of applications in protein engineering, proteins with existing utility have the potential for integrating improved or novel functionalities. The engineered haloalkane dehalogenase, a self-labeling protein tag derived from the haloalkane dehalogenase enzyme, is highly versatile and a valuable component for protein analysis technology development, such as clinical therapeutics and diagnostics. Rather than using randomized mutations to achieve the desired properties of the engineered haloalkane dehalogenase protein, ASR computes various combinations of feasible sequences that can then be tested and screened for functionality, stability, and changeable properties for fluorescent substrates. However, there are many uncertainties about the engineered haloalkane dehalogenase such as, the presence of other residues that affect the interactions of the HaloTag-HaloLigand system, and whether the functionalities are dependent on the engineering of the Ligand or the engineering of the protein itself. To combat these obstacles, ancestral sequence reconstruction was utilized to reveal the evolutionary trajectory of haloalkane dehalogenase (HLD-II) enzymes, displaying the conserved and amenable regions of predicted ancestors.

Mentor

Daniel Woldring