Repository logo

Application of PI-deconvolution to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assay

dc.contributor.advisorGeyer, Ronen_US
dc.contributor.committeeMemberDmitriev, Olegen_US
dc.contributor.committeeMemberWarrington, Roberten_US
dc.contributor.committeeMemberLin, Yen-Hanen_US
dc.contributor.committeeMemberKhandelwal, Ramjien_US
dc.contributor.committeeMemberDeCoteau, Johnen_US
dc.creatorAparicio de Navaraez, Albertoen_US 2008en_US
dc.description.abstractReagents that bind proteins are applicable in biology for detection of molecules, perturbation of signaling pathways and development of small-molecule pharmaceuticals. Protein ligands interact with proteins, inhibiting or altering their function. They are isolated from combinatorial libraries to interact with a specific target, using selection techniques such as phage display or yeast-two-hybrid assay. For the latter, one inconvenience is the detection of false positives, which can be solved by screening pools containing the samples to be tested, instead of individual samples. Samples are distributed in the pools following a pooling design. The PI-deconvolution pooling design was developed to screen cDNA libraries using the yeast-two-hybrid assay, which are smaller in size than protein ligand combinatorial libraries. Modifications to the PI-deconvolution screening technique were developed to adapt it to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assay. Every spot of the array containing the combinatorial library was randomly pooled. However, the yeast-two-hybrid assay loses sensitivity when strains are pooled. As PI-deconvolution requires detecting every interaction, we determined the optimal amount of library members that can be pooled in a spot, and the optimal number of replicates to ensure the detection of an interaction. The yeast-two-hybrid assay was used to perform a screening of a combinatorial library with seven domains of BCR-ABL, which were pooled according to PI-deconvolution. BCR-ABL is a chimeric protein with unregulated kinase activity that is responsible for chronic myelogenous leukemia. The scaffold used in the combinatorial library was an engineered intein that forms lariat peptides. After a screening of this library was performed, positive interactions were detected in 775 spots of the arrays that contained 1432 positive hits. Only 53 spots were deconvoluted. The coding sequences of the lariat peptides were determined for 23 lariat peptides interacted with the GEF domain of BCR, and for ABL, two with the FABD domain, one with the SH1 domain, and one with the SH3 domain. Finally, a β-galactosidase assay was performed to assess the affinity of the lariat peptides for their target. The isolated lariat peptides are potential inhibitors of BCR-ABL that can have therapeutic potential. This study will improve other screenings of combinatorial libraries with the yeast-two-hybrid assay.en_US
dc.subjectcombinatorial chemistryen_US
dc.subjectyeast-two-hybrid assayen_US
dc.subjectchronic myelogenous leukemiaen_US
dc.subjectbcr-abl inhibitoren_US
dc.titleApplication of PI-deconvolution to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assayen_US
dc.type.materialtexten_US of Saskatchewanen_US of Science (M.Sc.)en_US


Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
2.03 MB
Adobe Portable Document Format
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
905 B
Plain Text