Abstract
Kinases represent a vast and varied group of proteins, having more than 500 members in human beings alone. They have been found to be involved in various signal transduction pathways, ranging from embryonic development to apoptosis. The vaccinia-related kinases (VRKs) are a small sub-group of kinases containing three members: VRK1, VRK2, and VRK3. These proteins are involved in the cell cycle, cellular proliferation, chromatin condensation and gene transcription. VRK1 has been extensively studied not only for its normal biological roles, but also for its link to cancer. VRK2 is not as well studied as VRK1. However, the two kinases are quite closely related, sharing as much as 56% of their amino acid sequence. This indicates that there may be similarities not only in their function, but also in their influence on biological processes. Further research into VRK2 will be required to better understand its structure, function and influence in the context of normal cellular behavior, but also in the context of disease.
For this project, we have transformed bacteria with a plasmid containing our VRK2 sequence with a His-tag. The protein was extracted from these cells and purified by filtration and affinity chromatography. The purified protein was run on an SDS-PAGE to verify the presence of VRK2, and the absence of contaminant protein. Concentration was determined via Bradford assay. Finally, VRK2 was subjected to circular dichroism and differential scanning fluorimetry for specific characterization. The SDS-PAGE results showed that we were able to produce and purify our target protein, and the Bradford assay showed it could be produced at relatively high concentrations. Through DSF and CD we were able to experimentally determine melting point temperatures of VRK2, with and without ADP bound.
College
College of Science & Engineering
Department
Chemistry
Campus
Winona
First Advisor/Mentor
Emily Ruff
Start Date
4-19-2023 10:00 AM
End Date
4-19-2023 11:00 AM
Presentation Type
Poster Session
Format of Presentation or Performance
In-Person
Session
1b=10am-11am
Poster Number
39
Included in
Purification and Characterization of VRK2 for Further Research
Kinases represent a vast and varied group of proteins, having more than 500 members in human beings alone. They have been found to be involved in various signal transduction pathways, ranging from embryonic development to apoptosis. The vaccinia-related kinases (VRKs) are a small sub-group of kinases containing three members: VRK1, VRK2, and VRK3. These proteins are involved in the cell cycle, cellular proliferation, chromatin condensation and gene transcription. VRK1 has been extensively studied not only for its normal biological roles, but also for its link to cancer. VRK2 is not as well studied as VRK1. However, the two kinases are quite closely related, sharing as much as 56% of their amino acid sequence. This indicates that there may be similarities not only in their function, but also in their influence on biological processes. Further research into VRK2 will be required to better understand its structure, function and influence in the context of normal cellular behavior, but also in the context of disease.
For this project, we have transformed bacteria with a plasmid containing our VRK2 sequence with a His-tag. The protein was extracted from these cells and purified by filtration and affinity chromatography. The purified protein was run on an SDS-PAGE to verify the presence of VRK2, and the absence of contaminant protein. Concentration was determined via Bradford assay. Finally, VRK2 was subjected to circular dichroism and differential scanning fluorimetry for specific characterization. The SDS-PAGE results showed that we were able to produce and purify our target protein, and the Bradford assay showed it could be produced at relatively high concentrations. Through DSF and CD we were able to experimentally determine melting point temperatures of VRK2, with and without ADP bound.
