Yeast 2 Hybrid System to Find Map Kinase (MK-2) Interacting Binding Proteins
Disciplines
Medicine and Health Sciences
Abstract (300 words maximum)
The protein MAPKAPK2 (MK2) encodes for Ser/Thr in the protein kinase family, and along with p38 MAP kinase, play a crucial role in many cellular processes including inflammatory responses, nuclear export, gene expression regulation, and cell proliferation. The goal of this C.U.R.E.(course-based undergraduate research experiment) study is to find out more information about the MK2 protein, its function, and possible therapeutics in cancer and medicine research. To achieve this, a Yeast 2 Hybrid (Y2H) assay was utilized to screen for proteins interacting with MK2. The Yeast 2 Hybrid assay involved the insertion of a Gal4 Binding Domain (BD) fused with a bait protein and Gal4 Activation Domain (AD) fused with a prey protein. If the bait and prey proteins interact, GAL4 transcription factor is reconstituted and the reporter genes will be turned on, one of which, the alpha galactosidase, will turn the yeast colonies blue. The autoactivation of Gal4 by the “bait” protein, MK2, was assessed along with the positive and negative controls. The positive control was p53 with a BD and a T-antigen with a AD, these are known interactors and, in the Yeast 2 Hybrid system, did turn the yeast colonies blue. The negative control was Lamin with a BD (pGBKT7-LAM) and a T-antigen with an AD, these plasmids do not interact and no blue colonies were observed. The positive control of the reporter genes exhibited activation, while the negative control exhibited no activation. The experimental findings align with the predicted plates where interactions were expected to occur; similarly, the plates where no interactions were anticipated did align with the predictions. The Yeast 2 Hybrid System further supported the notion that the binding domain cannot activate the reporter genes by itself.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Rajnish Singh
Yeast 2 Hybrid System to Find Map Kinase (MK-2) Interacting Binding Proteins
The protein MAPKAPK2 (MK2) encodes for Ser/Thr in the protein kinase family, and along with p38 MAP kinase, play a crucial role in many cellular processes including inflammatory responses, nuclear export, gene expression regulation, and cell proliferation. The goal of this C.U.R.E.(course-based undergraduate research experiment) study is to find out more information about the MK2 protein, its function, and possible therapeutics in cancer and medicine research. To achieve this, a Yeast 2 Hybrid (Y2H) assay was utilized to screen for proteins interacting with MK2. The Yeast 2 Hybrid assay involved the insertion of a Gal4 Binding Domain (BD) fused with a bait protein and Gal4 Activation Domain (AD) fused with a prey protein. If the bait and prey proteins interact, GAL4 transcription factor is reconstituted and the reporter genes will be turned on, one of which, the alpha galactosidase, will turn the yeast colonies blue. The autoactivation of Gal4 by the “bait” protein, MK2, was assessed along with the positive and negative controls. The positive control was p53 with a BD and a T-antigen with a AD, these are known interactors and, in the Yeast 2 Hybrid system, did turn the yeast colonies blue. The negative control was Lamin with a BD (pGBKT7-LAM) and a T-antigen with an AD, these plasmids do not interact and no blue colonies were observed. The positive control of the reporter genes exhibited activation, while the negative control exhibited no activation. The experimental findings align with the predicted plates where interactions were expected to occur; similarly, the plates where no interactions were anticipated did align with the predictions. The Yeast 2 Hybrid System further supported the notion that the binding domain cannot activate the reporter genes by itself.