Screening of MK2 Interacting Proteins in Human cDNA Libraries Using a Yeast Two-Hybrid Assay
Disciplines
Biochemistry
Abstract (300 words maximum)
Mitogen-activated-protein-kinase-activated-protein-kinase 2 (MK2) is a kinase that phosphorylates substrates. The short isoform is of interest to our Course-Based Undergraduate Research Experience (CURE) CHEM 3512L class. This project seeks to better understand MK2’s function by finding its interacting/binding partners. To facilitate this, the Matchmaker Gold Yeast Two-Hybrid Assay was used. This method is based on the reconstitution of a transcriptional activator through the “bait” and “prey” proteins. The “bait” protein, MK2, is cloned into a binding vector to express a protein tagged with the binding domain. This protein is then screened against a library of “prey” proteins tagged with an activation domain. If the bait and prey proteins interact, then the binding and activation domains will come together and activate the Gal4 transcription factor resulting in the transcription of four reporter genes, AURI-C, HIS3, ADE1, and MEL1. Autoactivation of MK2-binding domain fusion protein was tested by screening for the expression of Gal4 reporter genes, namely aureobascidin resistance and alpha-galactosidase activity. No autoactivation of reporter genes in yeast Y190 transformed only with the binding domain-MK2 plasmid was observed, as evident by no growth on aureobasidin-containing plates and no blue colonies on X-alpha gal-containing plates. A positive control experiment was implemented by testing known interactors, namely p53 with the binding domain and T-antigen with the activation domain. Since these proteins interact in yeast, blue colonies were obtained, and colonies grew on -leu-trp/X-alpha-GAL/AbA plates. Negative controls were performed with laminin with the binding domain and T-antigen with the activation domain. No colonies grew on the aureobasicin plates, and no blue colonies were detected on the double dropout plates. Next steps involve transforming Y190 yeast cells with MK2-binding domain plasmid and mating them with Y187 yeast cells containing a cDNA library of human proteins to screen for potential binding partners through the yeast two-hybrid assay.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Rajnish Singh
Screening of MK2 Interacting Proteins in Human cDNA Libraries Using a Yeast Two-Hybrid Assay
Mitogen-activated-protein-kinase-activated-protein-kinase 2 (MK2) is a kinase that phosphorylates substrates. The short isoform is of interest to our Course-Based Undergraduate Research Experience (CURE) CHEM 3512L class. This project seeks to better understand MK2’s function by finding its interacting/binding partners. To facilitate this, the Matchmaker Gold Yeast Two-Hybrid Assay was used. This method is based on the reconstitution of a transcriptional activator through the “bait” and “prey” proteins. The “bait” protein, MK2, is cloned into a binding vector to express a protein tagged with the binding domain. This protein is then screened against a library of “prey” proteins tagged with an activation domain. If the bait and prey proteins interact, then the binding and activation domains will come together and activate the Gal4 transcription factor resulting in the transcription of four reporter genes, AURI-C, HIS3, ADE1, and MEL1. Autoactivation of MK2-binding domain fusion protein was tested by screening for the expression of Gal4 reporter genes, namely aureobascidin resistance and alpha-galactosidase activity. No autoactivation of reporter genes in yeast Y190 transformed only with the binding domain-MK2 plasmid was observed, as evident by no growth on aureobasidin-containing plates and no blue colonies on X-alpha gal-containing plates. A positive control experiment was implemented by testing known interactors, namely p53 with the binding domain and T-antigen with the activation domain. Since these proteins interact in yeast, blue colonies were obtained, and colonies grew on -leu-trp/X-alpha-GAL/AbA plates. Negative controls were performed with laminin with the binding domain and T-antigen with the activation domain. No colonies grew on the aureobasicin plates, and no blue colonies were detected on the double dropout plates. Next steps involve transforming Y190 yeast cells with MK2-binding domain plasmid and mating them with Y187 yeast cells containing a cDNA library of human proteins to screen for potential binding partners through the yeast two-hybrid assay.