DEVELOPMENT OF SPLIT-PROTEIN REASSEMBLY TOOLS TO STUDY KINASE FUNCTION
AuthorRounseville, Benjamin James
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PublisherThe University of Arizona.
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AbstractTo better study the role of a specific kinase and bypass the well-known challenge of selectively targeting kinases, we designed a ligand-gated split-protein system where two halves of a kinase are reassembled via the addition of a Chemical Inducer of Dimerization (CID) with concomitant activation of the enzyme. This was done by splitting the catalytic domain of tyrosine kinase Src into two inactive halves, and linking each to a copy of E. coli dihydrofolate reductase (eDHFR). Trimethoprim (TMP) is a tight and selective ligand for eDHFR, and in the presence of a di-TMP ligand, the previously mentioned Src-eDHFR halves dimerize, achieving reassembly into a functional kinase with measureable activity. To demonstrate the use of our Src-eDHFR system in cellulo, we expressed the splitkinase in mammalian cells and measured overall phosphorylation via western blotting. We have shown that the activity of our Src-eDHFR system is dependent on the presence of the di-TMP ligand. In addition, we have shown that two well identified mutations (K298M and T341M) behave the same way in our split system as they do in the native, unaltered protein. Overall, our eDHFR-Src system seems able to replicate the function of Src in cellulo, giving future researchers a useful tool to elucidate the role Src plays in different cellular processes.
Degree ProgramHonors College