Applications of Engineered Proteins in Redox Biology and Biomarker Detection Assay Development

Engineered proteins are very versatile tools that have been applied in assay development for various purposes. They have been made into genetically encoded biosensors/probes or affinity agents for biomarker detection. This thesis explored a few topics using assays developed with engineered proteins. The genetically encoded hydrogen peroxide generator, D-amino acid oxidase (DAAO), was used to understand the hours-long intracellular hydrogen peroxide (H₂O₂) generation. This study elucidated that the primary respondent of cytosolic H₂O₂ is peroxiredoxin 1 and the H₂O₂ induced apoptosis initiates before the collapse of Prx/Trx/TR antioxidant network. Then, a genetically encoded FRET sensor was used to design a high-throughput screening assay that identified three small-molecule drugs from over 600 compounds that can mediate toxicity through H₂O₂. This thesis also explored the applications of engineered proteins in diagnostic assay development. I engineered binders against various targets for gram-positive and gram-negative pathogenic bacteria, and two of them that have been tested and showed binding to Salmonella whole cells. The engineered binders were also used to develop a SARS-CoV-2 rapid tests. In this project, sikes lab members developed a paper-based assay to detect the SARS-CoV-2 nucleocapsid protein as a team and successfully validated the assay with patient samples. Subsequently, I improved the thermo-stability of the reporter binder protein used in the assay by switching the fusion partner of the binder to a thermally stable protein. I also identified the bottleneck of an epigentotyping assay development and provided insight for future direction.

Name thesis:degree_name

Doctoral

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemical Engineering

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2022

Author dc:creator

• Hao, Yining

Advisor dc:contributor.advisor

• Sikes, Hadley D.

Statement dc:rights

• In Copyright - Educational Use Permitted
• Copyright MIT

Licence dc:rights.uri

• http://rightsstatements.org/page/InC-EDU/1.0/

Handle dc:identifier.uri

https://hdl.handle.net/1721.1/145060

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/145060