Cartographie des complexes multiprotéiques humains suite à la modification ciblée du génome

Authors: Loehr, Jérémy
Advisor: Doyon, Yannick
Abstract: Conventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method to decipher molecular interaction networks and infer protein function. However, it is sensitive to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and ZFNs to insert cDNA of interest in the endogenous genomic safe harbor locus AAVS1 and purified several DNA repair and chromatin modifying holoenzymes to near homogeneity. We uncovered novel subunits and interactions amongst well-characterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small and large-scale explorations of protein interactions, as well as the study of biochemical activities and structure-function relationships.
Document Type: Mémoire de maîtrise
Issue Date: 2017
Open Access Date: 24 April 2018
Grantor: Université Laval
Collection:Thèses et mémoires

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