Date of Award
2024
Document Type
Thesis
Degree Name
Master of Science (M.S.)
Department
Pharmaceutical and Chemical Sciences
First Advisor
Mamoun M. Alhamadsheh
First Committee Member
William K. Chan
Second Committee Member
Jianhua Ren
Abstract
Nanobodies, also known as single-domain antibodies, are the variable heavy chain fragments of heavy chain-only antibodies with a molecular weight of ~15 kDa. Antibodies and nanobodies can selectively bind to a specific antigen; however, nanobodies have approximately 1/10th the molecular weight of antibodies. Nanobodies have been commonly used for stabilizing proteins in x-ray crystallography studies. More recently, nanobodies have been used for therapeutic purposes. In 2019, the FDA approved the first nanobody-based therapeutic. The myriad benefits of nanobodies include their stability in varying environments, ease of manufacturing, and good tumor penetration due to their smaller size. However, due to their small size, nanobodies suffer from a short half-life.Previously, our laboratory designed and developed novel methods to increase the half-life of small molecules, peptides, and proteins. Utilizing the past successes in developing half-life extension strategies, the ultimate goal of this study was to design novel ways to increase the half-life of nanobody therapeutics. We plan to use an anti-HER2 nanobody as a proof of concept. In this work, we report on the optimization processes in expressing and purifying the anti-HER2 nanobody. While expressing nanobodies in the periplasm using a BL21(DE3) E. coli host proved to be difficult, the most optimized protein expression method in the cytoplasm utilized a Rosetta-gami B(DE3) pLysS E. coli host with induction at 0.25 mM IPTG, 225 rpm, and 18°C for 16 hours.
Pages
51
Recommended Citation
Ho, Joshua S.. (2024). Enhancing the Expression and Purification of Anti-HER2 Nanobodies. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/4306
Included in
Biochemistry Commons, Biology Commons, Medicine and Health Sciences Commons, Molecular Biology Commons
Rights Statement
In Copyright. URI: http://rightsstatements.org/vocab/InC/1.0/
This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
