Assoc ProfessorDavid O'Connell

Effective exploitation of the proteome for biotechnological applications requires continued innovation in expression and extraction of highly-heterogenous protein classes from cellular systems, and their rigorous structure and function characterization. We address this need by developing a fragment complementation system that directs protein assembly in a toolbox for responsive biomaterials, sensors, and cryo-EM enabling advances. A particular area requiring systematic improvement is cryo-EM structure imaging and analysis, where a universal inert tool for structure detection and determination is a significant unmet need. We have identified a highly attractive protein scaffold, S100G, to build switchable biomaterials and sensors and through split-tag purification and immobilization strategies, effectively lower the barrier to cryo-EM structural analysis. We will build a range of biotechnological applications based on the responsiveness of S100G reconstitution from its two EF-hand subdomains to mild changes in solution conditions. A universal inert tool for cryo-EM includes one EF-hand on the protein-of-interest and the other EF-hand on an easily recognizable element. The Ca 2+ -switchable biomaterials provide systems for controlled formation and disassembly in cargo encapsulation and controlled release. The Ca 2+ sensor provides enhanced stringency and sensitivity and serve as a proof-of-concept for more challenging sensors for small molecules, akin to the portable glucose sensors with wide applicability in diabetes. We will develop powerful new tools for the purification of difficult targets, including membrane proteins, and improved and renewable sensor chip immobilization strategies. To facilitate industrialization of our tools, we will refine and scale- up the production and purification towards the kg batch scale. With these designs and their optimized production, we aim for sustainable and scalable solutions to meet important challenges in industrial biotechnology.