Date of Award

6-2021

Document Type

Thesis

Publisher

Santa Clara : Santa Clara University, 2021.

Department

Bioengineering

First Advisor

Zhiwen Zhang

Abstract

Protein ligation is a process integral to many biomedical applications, however the current industry standard, expressed protein ligation (EPL), is highly error prone and time-and-cost inefficient. Our team sought to improve this integral process by proving the efficacy of a new method of protein ligation using the transpeptidase Sortase A. Sortase A is known to efficiently bind two peptide fragments so long as they are attached to LPXTG motifs, which signal and activate the enzyme’s ligation abilities. Previous studies affirm Sortase A’s binding ability and efficacy in in vitro situations, however none have yet confirmed its abilities in vivo. Operating in vivo would not only allow us to take advantage of Sortase A’s binding abilities, but also the mammalian expression system’s own protein editing mechanisms, namely its high fidelity protein synthesis and post translational modification capabilities. This proof-of-concept project used Green Fluorescent Protein (GFP) fragments as a simple but qualitative method of measuring Sortase A’s in vivo binding abilities, causing the cells to fluoresce if the introduced GFP fragments were successfully bound together. Our team found that Sortase A does in fact have the capacity to operate in vivo, signified by fluorescence in our test subject and further confirmed by our positive and negative controls. These findings indicate a bright future for Sortase A’s role as a protein ligate, and a basis for future studies on how to further optimize and regulate its abilities to further improve protein ligation and its many applications.

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