Date on Senior Honors Thesis

5-2025

Document Type

Senior Honors Thesis

Degree Name

B.A.

Cooperating University

University of Louisville

Department

Biology

Degree Program

College of Arts and Sciences

Committee Chair

Richard Lamont

Committee Member

Deborah Yoder-Himes

Committee Member

Cara Cashon

Author's Keywords

Porphyromonas gingivalis Tyrosine Kinase 1, Post-Translational Modification, Ptk1, Porphyromonas gingivalis, protein purification

Abstract

The goal of this study was to optimize the expression and purification of Porphyromonas gingivalis Tyrosine Kinase 1 (Ptk1) in recombinant Escherichia coli containing the plasmids Gro_ESL and Ptk1, with a His6 tag, to obtain a highly pure protein sample. Various experimental conditions were tested to determine the most effective parameters for protein expression and purification, including adjustments in induction timing, buffer composition, and purification techniques.

Optimal protein expression was achieved when cells were induced at an optical density (OD) of 0.3 to 0.4. β-D-1-thiogalactopyranoside (IPTG), which activates the lac operon, was used to induce Ptk1 expression. A concentration of both 0.25 and 0.5 mM IPTG were found to be sufficient for maximizing expression while minimizing unintended protein production.

Purification was carried out using immobilized metal ion affinity chromatography (MIAC), which selectively binds the His6-tagged Ptk1. Wash buffers containing imidazole at concentrations between 20 and 35 mM were tested to improve purification efficiency. While these trials resulted in a significantly purer product, SDS-PAGE electrophoresis with Coomassie staining identified two to three contaminant proteins persisting across all trials.

This study made significant progress in identifying optimal techniques for future work, despite some possible impurities in the product. Further testing is needed to quantify the purity of the product. This study provides a strong foundation for refining protein purification strategies and improving yield, ultimately advancing the study of Ptk1 function and its role in P. gingivalis pathogenesis.

Lay Summary

Periodontal disease is a destructive condition that damages the periodontium, the tissues that support the teeth. The mildest form, gingivitis, affects around 90% of the population at some point in their lifetime, while severe periodontal disease impacts approximately 1.1 billion people worldwide, making it one of the most common chronic inflammatory diseases. A key contributor to this disease is Porphyromonas gingivalis (P. gingivalis), a bacterium that disrupts the host’s immune response and accelerates tissue damage. A P. gingivalis protein, Ptk1, enhances the bacterium’s ability to harm the host. However, research on Ptk1 remains limited, and further studies are needed to understand its role in disease progression.

To facilitate future research, this study aimed to produce a purified sample of the Ptk1 protein. Most contaminants were successfully removed, and experimental data provided insights into optimizing the process. The study involved two major steps: protein production and purification. To generate Ptk1, recombinant E. coli bacteria containing plasmid sequencing from P. gingivalis were cultivated in nutrient-rich broth with oxygen, and protein production was carefully controlled by adjusting the concentration of an inducing molecule to prevent overproduction, which can harm the bacterial host.

For purification, affinity chromatography was used, a technique that separates proteins based on their ability to bind to a specific tag. In this experiment, the Ptk1 protein was engineered with a histidine tag (His-tag), a small sequence of amino acids that allows it to bind to a chromatography column containing nickel. Imidazole, a molecule that mimics histidine, was then introduced to compete with the His-tag and displace the protein from the column. The concentration of imidazole played a crucial role in purification—too little imidazole could leave unwanted proteins bound to the column, while too much could wash out impurities along with Ptk1. By testing different concentrations, this study identified potential optimizations for producing Ptk1 more effectively. This process will enable deeper investigation into Ptk1’s functions, ultimately contributing to the development of targeted treatments for periodontal disease.

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