Screening of MK2 Interacting Proteins in Human cDNA Libraries Using a Yeast Two-Hybrid Assay
Disciplines
Biochemistry | Life Sciences
Abstract (300 words maximum)
Mitogen-activated-protein-kinase-activated-protein-kinase 2 (MK2) is a kinase that phosphorylates substrates. This project seeks to better understand the function of MK2’s short isoform by finding its interacting/binding partners. The Matchmaker Gold Yeast Two-Hybrid Assay was used since it depends on the reconstitution of a transcriptional activator through “bait” and “prey” proteins. The “bait” protein, MK2, was cloned into a binding vector to express a protein tagged with the binding domain. It was screened against a library of “prey” proteins tagged with an activation domain. If these proteins interact, the binding and activation domains come together and activate the Gal4 transcription factor resulting in the transcription of reporter genes AURI-C, HIS3, ADE1, and MEL1. By screening for the expression of these genes, experiments confirmed that the MK2-binding domain fusion protein doesn’t autoactivate the transcription factor as evidenced by no growth on aureobasidin-containing plates and no blue colonies on X-alpha gal-containing plates. A positive control experiment used p53 with the binding domain, and T-antigen with the activation domain, resulting in blue colonies 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, resulting in no growth/blue colonies on the aureobasidin and double dropout plates respectively. Y190 yeast cells were transformed with MK2-binding domain plasmid and mated with Y187 yeast cells containing a cDNA library of human proteins to screen for potential binding partners. Approximately 200 positives were obtained and stored in a solution of glycerol and YPDA. Ten positives were analyzed. PCR amplification of cDNA inserts from positive colonies revealed that the “prey” plasmids were non-identical. The plasmids were isolated from yeast cells, transformed into DH5α E. coli, and plated on LB-amp plates to select the ampicillin-resistant “prey” plasmids from which it was isolated. The next steps involve characterizing the cDNA inserts by sequencing.
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. This project seeks to better understand the function of MK2’s short isoform by finding its interacting/binding partners. The Matchmaker Gold Yeast Two-Hybrid Assay was used since it depends on the reconstitution of a transcriptional activator through “bait” and “prey” proteins. The “bait” protein, MK2, was cloned into a binding vector to express a protein tagged with the binding domain. It was screened against a library of “prey” proteins tagged with an activation domain. If these proteins interact, the binding and activation domains come together and activate the Gal4 transcription factor resulting in the transcription of reporter genes AURI-C, HIS3, ADE1, and MEL1. By screening for the expression of these genes, experiments confirmed that the MK2-binding domain fusion protein doesn’t autoactivate the transcription factor as evidenced by no growth on aureobasidin-containing plates and no blue colonies on X-alpha gal-containing plates. A positive control experiment used p53 with the binding domain, and T-antigen with the activation domain, resulting in blue colonies 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, resulting in no growth/blue colonies on the aureobasidin and double dropout plates respectively. Y190 yeast cells were transformed with MK2-binding domain plasmid and mated with Y187 yeast cells containing a cDNA library of human proteins to screen for potential binding partners. Approximately 200 positives were obtained and stored in a solution of glycerol and YPDA. Ten positives were analyzed. PCR amplification of cDNA inserts from positive colonies revealed that the “prey” plasmids were non-identical. The plasmids were isolated from yeast cells, transformed into DH5α E. coli, and plated on LB-amp plates to select the ampicillin-resistant “prey” plasmids from which it was isolated. The next steps involve characterizing the cDNA inserts by sequencing.