Project Director

Harris, Bradley Jordan

Department Examiner

Wigal, Cecelia; Turgeson, Andrew

Department

Dept. of Civil and Chemical Engineering

Publisher

University of Tennessee at Chattanooga

Place of Publication

Chattanooga (Tenn.)

Abstract

Green fluorescent protein, GFP, is a widely used fluorescent reporter, and its purification provides a clear model for studying downstream bioprocessing. This thesis investigates how binding-buffer salt strength affects GFP retention and elution during hydrophobic interaction chromatography, HIC, by comparing three conditions (1.0×, 0.8×, and 0.6×). GFP-expressing E. coli were lysed and clarified, and the lysate was purified on HIC columns; collected fractions were evaluated qualitatively under UV illumination to track fluorescence distribution. Results showed a salt-dependent trend in which higher salt improved retention and shifted fluorescence toward later elution fractions, while reduced salt weakened hydrophobic interactions and increased earlier release. These findings demonstrate how a single controllable parameter can tune HIC separation behavior and will be used to develop a graduate-level protein purification laboratory protocol at the University of Tennessee at Chattanooga.

Acknowledgments

I would like to thank my advisor, Dr. Bradley Harris, for the time and dedication he has invested in me over the past several years, even before I began this research. His mentorship has been instrumental in my college experience and in shaping the engineer I am becoming. I am also grateful to my committee members, Dr. Cecelia Wigal and Dr. Andrew Turgeson, for their guidance and support throughout this project. Dr. Wigal, in particular, has encouraged me to think about engineering in a broader context and has been a steady source of encouragement during my time at UTC. I would also like to thank my family for their unwavering support and for providing me with opportunities that I will always be grateful for. Finally, thank you to my cat, Tinny, for keeping me company through every late night of research and writing.

Degree

B. S.; An honors thesis submitted to the faculty of the University of Tennessee at Chattanooga in partial fulfillment of the requirements of the degree of Bachelor of Science.

Date

5-2026

Subject

Green fluorescent proteins--Purification; Chromatography, Hydrophobic interaction; Proteins--Purification

Keyword

Green fluorescent protein; hydrophobic interaction chromatography; salt effects; UV fluorescence

Discipline

Chemical Engineering

Document Type

Theses

Extent

40 unnumbered leaves

DCMI Type

Text

Language

English

Rights

http://rightsstatements.org/vocab/InC/1.0/

License

http://creativecommons.org/licenses/by/4.0/

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