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Publication Date

2022-05-09

First Advisor

Sarah J. Moore

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Engineering

Keywords

protein engineering, mesothelin, cancer, targeted drug delivery, non-canonical amino acid

Abstract

Though overall cancer mortality rates have been decreasing in the last two decades, current cancer treatments still come with a wide range of side effects that can be detrimental to a patient’s quality of life. There is a critical need for cancer treatment solutions like precision medicine, which allows for the development of treatments based on the molecular and genetic characteristics of a disease to increase treatment efficacy and lower toxicity. Targeted drug delivery systems, for instance, aim to carry drug molecules directly to cancer cells by targeting biomarkers with a binding protein. Studies have shown that the cell surface receptor mesothelin (MSLN) is abundantly expressed in multiple types of cancer, and is thus an attractive biomarker for targeted drug delivery. Previous members of the Moore lab have engineered non-antibody fibronectin type III (Fn3) proteins which have been shown to selectively bind to MSLN receptors. Now, work must be done to enable conjugation of a drug or drug-loaded polymer to the Fn3 protein for a complete targeted drug delivery mechanism. The goal of this thesis was to work towards the incorporation of the non-canonical amino acid azidohomoalanine to allow for Fn3/polymer binding. Azidohomoalanine cannot be directly incorporated into a protein structure, so first a direct amino acid addition using the azidohomoalanine canonical analog methionine needed to be performed. The placement of the new amino acid in the Fn3 protein structure was designed, and a polymerase chain reaction synthesis was conducted to add the methionine codon to the Fn3 DNA sequence. Two transformations were performed, first to clone the DNA sample and then to express the Fn3 protein with methionine incorporated (Fn3-met). A concentrated and purified protein stock of Fn3-met was successfully produced, and was used to conduct an initial binding assay study that confirmed Fn3-met binds to MSLN even with the added amino acid. Future 3 work will include conducting further binding assays to quantify any changes in the Fn3/MSLN binding affinity with the methionine addition, and moving forward with the substitution of azidohomoalanine for methionine in the Fn3-met protein structure.

Rights

©2022 Helen Danielson. Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

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