Year of Award
2024
Document Type
Dissertation
Degree Type
Doctor of Philosophy (PhD)
Degree Name
Biochemistry & Biophysics
Department or School/College
Biochemistry and Biophysics Program
Committee Chair
Bruce Bowler
Commitee Members
Ekaterina Voronina, Elizabeth Putnam, Brent Ryckman, Travis Hughes
Abstract
Producing recombinant proteins stands as a cornerstone in both biotechnology and pharmaceutical research, supporting the creation of therapeutics, vaccines, and enzymes used in various industries. Bacterial systems for expression, especially Escherichia coli, are preferred due to their rapid growth rates, ease of genetic manipulation, and scalability. However, generating proteins under current Good Manufacturing Practice (cGMP) standards introduces substantial challenges, such as issues with scalability, toxicity, protein misfolding, and strict regulatory demands. This section delivers an in-depth review of these obstacles and current methods developed to address them. An extensive exploration is conducted into induction techniques, including auto-induction and isopropyl-D-1-thiogalactopyranoside (IPTG) induction. A detailed comparison of various inducers like galactose, lactose, and IPTG; emphasizes their individual strengths and limitations, particularly in the context of animal-free variants. Additionally, this analysis covers media formulations, specific case studies (including applications within the CAR-T cell field involving cGMP-grade CRISPR-Cas9 technology), and the distinct hurdles faced by the biotech industry compared to academia. Finally, a proposed pathway to navigate these challenges is outlined, highlighting recent innovations and strategies aimed at improving production capacity for downstream applications. Multiwavelength analytical ultracentrifugation (MW-AUC) serves as a cornerstone in our multi- method approach characterizing numerous samples, including those used in this study. In this current series of experiments, we provide a comprehensive strategy whereby we integrate MW- AUC with microscale thermophoresis (MST), isothermal titration calorimetry (ITC), and fluorescence correlation spectroscopy (FCS), in this way providing a holistic view of the thermodynamic and hydrodynamic properties of proteins under physiological solution-phase conditions. Each of these biophysical techniques contributes distinct, yet complementary insights, creating a robust and sophisticated analytical framework for accurate and thorough characterization of molecular interactions.
Recommended Citation
Bosco, James Gillette, "A Better Way to Make Protein" (2024). Graduate Student Theses, Dissertations, & Professional Papers. 12439.
https://scholarworks.umt.edu/etd/12439
© Copyright 2024 James Gillette Bosco
