Engineering and Characterizing Proteins: A Dual Study on Canine p53 Protein and Cyan Thermal Protein
Date of Graduation
Summer 2023
Degree
Master of Science in Chemistry
Department
Chemistry and Biochemistry
Committee Chair
Natasha DeVore
Abstract
This research aims to explore protein development and characterization, focusing on a dual study of canine p53 protein and cyan thermal protein. For the canine p53 protein, the goal is to comprehend its structure to better understand a key component of canine cancers. This multi-step process involved expressing the canine p53 DNA-binding domain in E. coli and purifying it through affinity and ion-exchange chromatography. The purified protein was studied to test the binding of canine p53 protein to human DNA sequences using Surface Plasmon Resonance (SPR) experiments and an Electrophoretic Mobility Shift Assay (EMSA), visualized with a SYBR-safe stain. The protein's three-dimensional structure is obtained via X-ray diffraction following the formation of protein crystals. Simultaneously, the study investigates the modification of the thermal green protein (TGP) into a cyan version by introducing a mutation at residue 67 that alters a tyrosine (Y) residue into a tryptophan (W) residue (Y67W) in the protein's chromophore. Successful mutation led to the chromophore fluorescing in the cyan region. The mutant protein was expressed in E. coli and purified using affinity and ion-exchange chromatography. The mutant's stability was determined through chemical, thermal, and pH stability assays. Secondary mutants were constructed, including a Q66E mutation or the I199 residue exchanged for either a serine (I199S) or a threonine (I199T) to stabilize the chromophore. Protein crystals were grown in the first step towards determining a crystal structure for CTP which may aid future efforts in optimizing the protein.
Keywords
canine p53 protein, cyan thermal protein, electrophoretic mobility shift assay, fluorescent, cancer
Subject Categories
Biochemistry
Copyright
© Dhruv B. Sitapara
Recommended Citation
Sitapara, Dhruv B., "Engineering and Characterizing Proteins: A Dual Study on Canine p53 Protein and Cyan Thermal Protein" (2023). MSU Graduate Theses. 3893.
https://bearworks.missouristate.edu/theses/3893
