Insertional Profiling of Pyrroloquinoline Quinone Glucose Dehydrogenase for Two-Component Biosensor Engineering

Abstract

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Currently, it is both expensive and invasive to screen for CRC. Developing a potential screening method that is more affordable and less invasive than endoscopy and biopsy would be beneficial to vulnerable communities. This method should also be able to diagnose and monitor the development and progression of GI cancers in real-time. A sensor that is capable of sending out an electrical signal in the presence of a cancer biomarker would alleviate the physical and economic costs of endoscopy while offering a significant amount of information about the gastrointestinal environment. Pyrroloquinoline quinone (PQQ) glucose dehydrogenase (PQQ-GDH), a redox enzyme, is capable of using glucose as a substrate for electron transfer. Engineering PQQ-GDH by insertion of a ligand-binding domain that is specific to a cancer biomarker would create an allosteric enzymatic switch that only produces current in the presence of the biomarker. To begin building this platform, I am creating a comprehensive insertion library to identify functionally important residues and domains of the PQQ-GDH. From there, I will profile which locations are capable of small primary, secondary, and/or tertiary structure disruptions. After identifying these regions, I will then create two-component protein switches capable of detecting cancer biomarkers. Creating an oxidoreductase switch that controls current transfer in the presence of gastrointestinal cancer biomarkers will advance the development of a novel, portable bio-recognition device capable of real-time, continuous sensing.