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

Citation

Li, Hanqing (2017) Development of a High-Throughput Protein Interaction Assay and its Applications. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9Q23X9R. http://resolver.caltech.edu/CaltechTHESIS:05292017-183651116

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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:protein interaction, nanomaterials for bone scaffolds
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Zinn, Kai George
Thesis Committee:
  • Sternberg, Paul W. (chair)
  • Fraser, Scott E.
  • Deshaies, Raymond Joseph
  • Zinn, Kai George
Defense Date:18 April 2017
Record Number:CaltechTHESIS:05292017-183651116
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:05292017-183651116
DOI:10.7907/Z9Q23X9R
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10217
Collection:CaltechTHESIS
Deposited By: Hanqing Li
Deposited On:06 Jun 2017 23:43
Last Modified:22 Feb 2018 23:32

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