Date of Graduation

Fall 2025

Degree

Master of Science in Chemistry

Department

Chemistry & Biochemistry

Committee Chair

Natasha DeVore

Abstract

This research investigated four different proteins: yellow thermal protein (YTP), blue thermal protein (BTP), lactoperoxidase (LPO), and thyroid peroxidase (TPO). YTP and BTP were both engineered from the extremely stable and non-aggregative thermal green protein (TGP) by mutating one residue each to obtain proteins with shifted spectra while preserving the stability properties observed in TGP. Site-directed mutagenesis (SDM) was utilized to construct YTP by mutating a histidine at residue 197 to a tyrosine (H197Y) and BTP by mutating a tyrosine at residue 67 to a histidine (Y67H). These proteins were expressed in E. Coli and purified with nickel and diethylaminoethyl (DEAE) column chromatography. UV/Vis and fluorescence spectra were obtained for YTP, BTP, and TGP. Preliminary chemostability measurements were run as well, and fluorescent lifetime data was obtained for BTP. Crystals of YTP and BTP were also obtained from conditions similar to that used to grow TGP crystals. Meanwhile, a plasmid containing a human LPO gene was designed and expressed in E. Coli cells with the end goal of obtaining active protein for use in crystallography. Seven expression and purification pairings took place, with purifications utilizing nickel and carboxymethyl (CM) column chromatography. UV/Vis spectra and SDS-PAGE were used to determine initial likelihood of expression success. Activity assays were performed several times, each time indicating no activity present. However, a western blot did detect LPO bands. Finally, a plasmid containing a truncated human TPO gene was also designed and expressed in E. Coli cells with the goal of obtaining active protein for use in crystallography. Purifications included nickel, DEAE, and size-exclusion column chromatography. Several expressions for use in western blots and crystal setups were performed, while two detergent types were also explored. In tandem, a TPO Course-Based Undergraduate Research Experience (CURE) lab was designed as an Intro to Biochemistry lab investigating six TPO mutations including the following techniques: SDM, protein purification, UV/Vis spectrophotometry, bicinchoninic acid (BCA) assay quantification, and a guaiacol assay.

Keywords

yellow thermal protein, blue thermal protein, lactoperoxidase, thyroid peroxidase, course-based undergraduate research experience

Subject Categories

Biochemistry

Copyright

© Cammi Dargatz

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Open Access

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Biochemistry Commons

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