Using a Yeast Two-Hybrid to study the protein interactions of mitogen‐activated protein kinase‐activated protein kinase‐2(MK2)
Disciplines
Biochemistry, Biophysics, and Structural Biology
Abstract (300 words maximum)
Using a Yeast Two-Hybrid to study the protein interactions of mitogen‐activated protein kinase‐activated protein kinase‐2(MK2)
Nathaniel Davis, Trinity Bell-Atkinson
These experiments were conducted as part of a CURE biochemistry lab, with 3-hour meetings once a week. Our protein of interest is the short isoform MK2, which is understudied compared to its long form. MK2 is significant because it plays a role in DNA transcription and regulation; learning about its functions could lead to clinical applications. By discovering what proteins MK2 interacts with, we can determine its function. We used yeast 2-hybrid screening to accomplish this. This method uses transcription factor GAL4 that binds to the GAL promoter and activates 4 reporter genes. This factor’s binding and activation domains can be separated and fused with bait protein and prey protein. When these fusion proteins are expressed in yeast, if the bait and prey protein interact, they reconstitute GAL 4 and activate the reporter genes. MK2 is our binding domain-bait fusion protein, and it will screen a library of prey human proteins. Before that, we performed an autoactivation assay on the binding domain. We used plasmids pGBKT7-53 and pGADT7-T-anitgen as positive controls because p53 interacts with T-antigen. We used plasmids pGBKT7-LAMININ and pGADT7-T-antigen as negative controls since Laminin and T-antigen are non-interactors. When our positive controls interact, they should secrete a-galactosidase enzyme, resulting in blue yeast colonies in the presence of substrate X-alpha-GAL. Our negative controls should not create colonies. When these were grown on agar plates, both results were observed. The final step is a cDNA library screen of human- activation domain fused proteins. When the MK2-BD successfully binds to a library protein, the resulting colonies will be blue because of interaction. By learning which proteins MK2 binds to, we can determine which cellular processes it is most likely to impact.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Rajnish Singh
Using a Yeast Two-Hybrid to study the protein interactions of mitogen‐activated protein kinase‐activated protein kinase‐2(MK2)
Using a Yeast Two-Hybrid to study the protein interactions of mitogen‐activated protein kinase‐activated protein kinase‐2(MK2)
Nathaniel Davis, Trinity Bell-Atkinson
These experiments were conducted as part of a CURE biochemistry lab, with 3-hour meetings once a week. Our protein of interest is the short isoform MK2, which is understudied compared to its long form. MK2 is significant because it plays a role in DNA transcription and regulation; learning about its functions could lead to clinical applications. By discovering what proteins MK2 interacts with, we can determine its function. We used yeast 2-hybrid screening to accomplish this. This method uses transcription factor GAL4 that binds to the GAL promoter and activates 4 reporter genes. This factor’s binding and activation domains can be separated and fused with bait protein and prey protein. When these fusion proteins are expressed in yeast, if the bait and prey protein interact, they reconstitute GAL 4 and activate the reporter genes. MK2 is our binding domain-bait fusion protein, and it will screen a library of prey human proteins. Before that, we performed an autoactivation assay on the binding domain. We used plasmids pGBKT7-53 and pGADT7-T-anitgen as positive controls because p53 interacts with T-antigen. We used plasmids pGBKT7-LAMININ and pGADT7-T-antigen as negative controls since Laminin and T-antigen are non-interactors. When our positive controls interact, they should secrete a-galactosidase enzyme, resulting in blue yeast colonies in the presence of substrate X-alpha-GAL. Our negative controls should not create colonies. When these were grown on agar plates, both results were observed. The final step is a cDNA library screen of human- activation domain fused proteins. When the MK2-BD successfully binds to a library protein, the resulting colonies will be blue because of interaction. By learning which proteins MK2 binds to, we can determine which cellular processes it is most likely to impact.