Development of a High-Throughput Protein Interaction Assay and its Applications

Author: Li, Hanqing

Year: 2017

Degree: Dissertation (Ph.D.)

Advisor: Zinn, Kai George

Committee Members: Sternberg, Paul W.; Fraser, Scott E.; Deshaies, Raymond Joseph; Zinn, Kai George

Option: Biology

DOI: 10.7907/Z9Q23X9R

Abstract

Protein-protein interactions (PPIs) form the backbone for a vast array of biological processes in an organism, ranging from signal transduction to gene regulation to intercellular signaling. Therefore, mapping out protein interactomes has been a crucial and prolific area of scientific research. In recent years, much progress has been made in generating high throughput protein interaction data in a variety of organisms, including S. cerevisiae, C. elegans, and D. melanogaster, as well as in human cell culture. The strength of protein interactions varies widely, from almost irreversible assembly of complexes to highly transient interactions. Because of their diversity and complexity, a wide variety of methods have been used to study protein interactions. This includes such commonly-used assays like yeast two-hybrid, to mass spectrometry, ELISA, affinity pulldowns, etc.

Despite the plethora of assays and data sets generated for PPIs, interactions involving cell surface and secreted proteins (CSSPs) remains underrepresented in the results. This is due to the fact that CSSP interactions tend to have lower KDs (around the μM range), and are generally highly transient and difficult to perform using standard assays such as yeast two-hybrid. To circumvent these problems, we designed a high-throughput PPI assay with high sensitivity. To validate the effectiveness of the assay, we utilized it to probe for interactions among two families of Drosophila CSS proteins, the Beats and the Sides, to see if we could recapitulate known interactions and uncover new ones. We were able to recapitulate almost all of the known interactions, as well as discover three novel ones. Additionally, we also studied the expression patterns of members of the Beat and Side families in Drosophila embryos and larvae, as well as analyzed the effects of mutations of Side-VI and Beat-Vs in embryos.

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