Construction of molecular tools through protein excision and splicing

With the explosion of protein tools as popular platforms for discovery and therapeutics, we see greater need for regulator systems that work congruently within these frameworks, especially safe and effective tools that can be implemented in humans. To this end, we endeavor to create orthogonal, precise and flexible protein modulators that can be easily employed to control protein tools with little need to iterate design for novel contexts. Hepatitis C NS3 protease is employed as a stabilizable linker between protein domains, enabling control over protein localization with FDA approved anti-viral drugs. The power of this tool is demonstrated by controlling gene expression through the controlled tether and release of a transcription factor. Inteins have already been employed to modulate proteins in synthetic contexts, however we observe that natural systems lack the avenues of control necessary to make them indispensable. We employ existing protein tools to construct a system of modular protein association, as well as drug and light inducible schema that reveal gaps in our knowledge of how to repurpose inteins in vivo. Despite this, we use inteins in the construction of a novel cargo delivery platform based on the fusogenic properties of the viral envelope glycoprotein from Vesicular stomatitis virus (VSV-G). We confirm reduced tropism of cargo delivery based on an intein lock-and-key mechanism that has implications for both biosafety as well as targeted delivery in vivo of natively folded proteins to target cells.2024-05-23T00:00:00