Biotechnology Applications of Engineered Intrinsically Disordered Protein Condensates

Abstract

The spontaneous phase transition of biological macromolecules from a homogenous solution into a dense biomolecular condensate underpins a plethora of natural processes, including cellular modulation of gene expression, control over metabolic processes, and cellular communication. Tuning this biomolecular condensation behavior would allow researchers to precisely control the organization of cellular components for a myriad of biotechnological applications. Current strategies to create bespoke biomolecular condensates rely on a small library of proteins that naturally form condensates but which lack well-understood design rules that would allow researchers to precisely control their phase separation behavior. In contrast, Elastin-like polypeptides (ELPs) are a class of simple, polymer-like proteins with extremely well-defined design rules governing their in vitro phase separation behavior. The rules dictating the intracellular condensation behavior of ELPs, however, remain only lightly characterized. In this thesis, we demonstrate the utility of synthetic ELP condensates in controlling bacterial cell physiology and describe the design rules governing ELP behavior in mammalian cells. We first create ELP condensates in E. coli which enhance expression of a target gene. By fusing ELPs to an RNA-binding protein and incorporating its cognate recognition sequence into a target mRNA, we design ELPs which could bind a target mRNA and, upon phase transitioning, sequester the mRNA into a condensate. We show that in E. coli, the sequestration of a target mRNA enhances its translation, presenting a novel method of activating gene expression. We then generalize the system by showing that condensates formed using a variety of RNA-binding proteins, ELPs, and target mRNA structures consistently enhance protein translation from sequestered mRNA. We then establish design rules for creating ELP condensates in mammalian cells. We create a library of ELPs with a range of sizes and hydrophobicities and demonstrate that these ELPs form phase-separated condensates in mammalian cells. We next demonstrate that smaller ELPs are able to efficiently enter the cell nucleus, while large ELPs are excluded from the nucleus proportionally to their propensity to form condensates. Finally, we show that ELPs can be localized to various organelles including the Golgi body and around the nucleus, directed solely by their amino acid sequences. We then show, for the first time, that ELPs can be efficiently secreted from commercial mammalian cell platforms commonly used to express therapeutic proteins. We first characterize the design rules governing ELP secretion and demonstrate that ELP secretion is proportional to an ELPs’ propensity to phase separate in the cell’s endoplasmic reticulum. We then demonstrate the secretion and complete assembly of antibody-ELP fusions with a variety of biomolecule architectures and show that these molecules can be purified through the reversible formation and dissolution of antibody-ELP condensates. Finally, we develop a chromatography-free method of purifying un-modified antibodies directly from mammalian cell culture through the co-secretion of ELPs that bind and sequester antibodies into synthetic condensates. This work demonstrates the utility of ELP condensates as a biotechnology platform for controlling cell physiology and the development of novel therapeutics.