Controllable, Synthetic Membrane-Less Organelles From Recombinant Proteins

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Doctor of Philosophy (PhD)
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membrane-less organelles
phase separation
synthetic biology
Chemical Engineering
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Reed, Ellen

How to precisely control the self-assembly of biomolecules is an important problem in engineering. This thesis focuses on the self-assembly of biological proteins to create suprastructures including micelles and coacervates. Molecular biology techniques are used to engineer recombinant proteins that assemble into structures with designer functionality and tailored responsiveness. I describe the design of protein coacervates that respond to external stimuli including proteases, redox conditions, and light. Two engineered, recombinant proteins are reported in this work: a protein that self assembles into micelles and coacervates that can be controlled using redox conditions and a protein that self assembles into coacervates in response to proteases and to 405 nm light. The redox responsive system utilizes the naturally occurring plant protein oleosin. A variant of oleosin self assembles into spherical micelles at low concentrations and into coacervates at high concentrations. The propensity of this oleosin variant to form coacervates increases when a cysteine residue is added to the protein. Furthermore, the transition temperature for coacervate formation is precisely tuned by changing the location of the cysteine residue and by blending oleosin variants. The protease and light responsive systems utilize the C. elegans protein, LAF-1. A tandem repeat of the N-terminal, intrinsically disordered, domain of LAF-1, RGG was fused maltose binding protein. Maltose binding protein (MBP) solubilizes RGG preventing coacervate formation. A protease recognition site or a photocleavable protein was added between MBP and RGG. By adding a protease or exposing the system to 405 nm light, MBP was removed from RGG resulting in the formation of protein coacervates. The development of light activated coacervates without the use of continuous illumination allows for the assembly of organelles with superior spatial and temporal control.

Daniel A. Hammer
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