Utilizing a Gal4-Based Yeast Two-Hybrid Assay to Screen for Protein-protein Interactions Using the Short Isoform of MK2
Disciplines
Biochemistry
Abstract (300 words maximum)
Complex protein-protein interactions, which are the foundation of functioning biological systems, can be comprehensively analyzed using the Yeast Two-Hybrid (Y2H) screening method. This assay utilizes Gal4, a yeast-specific transcription factor that possesses both a DNA-binding domain (DBD) and an activation domain (AD); when these two domains interact, Gal4 promotes the expression of four separate reporter genes. For this study, a Y2H assay was used to analyze the short isoform of mitogen‐activated‐protein‐kinase‐activated‐protein‐kinase‐2 (MK2), an enzyme with little-known function. The DNA sequence that codes for MK2 was inserted into a Gal4-DBD plasmid vector, creating a protein fusion termed MK2-BD or the “bait”. A cDNA library with human proteins fused to Gal4-AD – the “prey” proteins – will eventually be used to screen for potential MK2 interactors. If the “bait”, MK2, interacts with a “prey protein”, Gal4 should be reconstituted, leading to the activation of the reporter genes. A maxiprep was conducted to amplify the bait plasmid. The BD-containing plasmids pGBT9, pGBKT7-53, pGBKT7-Lam, and MK2-BD were transformed into a Y190 yeast strain, while pGADT7-T was transformed into a Y187 strain. Transformation into yeast with pGBT9 was used to calculate the transformation efficiency and indicated that the cells were successfully transformed. To test the efficacy of the Y2H system, pGBKT7-53 and pGADT7-T were mated as a positive control; pGBKT7-Lam and pGADT7-T were mated as a negative control. A blue-white screening was used to determine whether proteins were interactors or non-interactors. MK2-BD was also plated by itself to test for autoactivation of Gal4-regulated genes. Ultimately, the controls showed that the Y2H system functions well. This system will ultimately be used to screen for proteins that interact with MK2, and it may aid in the elucidation of its function. All experiments were done within a CURE course, where students met for about three hours once a week.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Dr. Rajnish Singh
Utilizing a Gal4-Based Yeast Two-Hybrid Assay to Screen for Protein-protein Interactions Using the Short Isoform of MK2
Complex protein-protein interactions, which are the foundation of functioning biological systems, can be comprehensively analyzed using the Yeast Two-Hybrid (Y2H) screening method. This assay utilizes Gal4, a yeast-specific transcription factor that possesses both a DNA-binding domain (DBD) and an activation domain (AD); when these two domains interact, Gal4 promotes the expression of four separate reporter genes. For this study, a Y2H assay was used to analyze the short isoform of mitogen‐activated‐protein‐kinase‐activated‐protein‐kinase‐2 (MK2), an enzyme with little-known function. The DNA sequence that codes for MK2 was inserted into a Gal4-DBD plasmid vector, creating a protein fusion termed MK2-BD or the “bait”. A cDNA library with human proteins fused to Gal4-AD – the “prey” proteins – will eventually be used to screen for potential MK2 interactors. If the “bait”, MK2, interacts with a “prey protein”, Gal4 should be reconstituted, leading to the activation of the reporter genes. A maxiprep was conducted to amplify the bait plasmid. The BD-containing plasmids pGBT9, pGBKT7-53, pGBKT7-Lam, and MK2-BD were transformed into a Y190 yeast strain, while pGADT7-T was transformed into a Y187 strain. Transformation into yeast with pGBT9 was used to calculate the transformation efficiency and indicated that the cells were successfully transformed. To test the efficacy of the Y2H system, pGBKT7-53 and pGADT7-T were mated as a positive control; pGBKT7-Lam and pGADT7-T were mated as a negative control. A blue-white screening was used to determine whether proteins were interactors or non-interactors. MK2-BD was also plated by itself to test for autoactivation of Gal4-regulated genes. Ultimately, the controls showed that the Y2H system functions well. This system will ultimately be used to screen for proteins that interact with MK2, and it may aid in the elucidation of its function. All experiments were done within a CURE course, where students met for about three hours once a week.