Protein resurfacing to identify macromolecular assemblies

摘要

Protein engineering is an emerging discipline that dovetails modern molecular biology techniques with high-throughput screening, laboratory evolution technologies, and computational approaches to modify sequence, structure, and in some cases, function and properties of proteins. The ultimate goal is to develop new proteins with improved or designer functions for use in biotechnology, medicine and basic research. One way to engineer proteins is to change their solvent exposed regions through focused or random 'protein resurfacing'. Here, I describe several approaches towards the development of synthetic proteins with new properties and function, including resistance to aggregation, increased solubility, and potent and selective macromolecule recognition. The first part of this thesis describes the use of protein supercharging to develop a split-superpositive GFP reassembly assay that is more efficient, faster, and more robust than previously described variants, largely due to increased resistance to aggregation. The second part of this thesis describes the use of shape complementarity, protein resurfacing, and high-throughput screening to evolve the first potent and selective protein-based inhibitor of the oncoprotein gankyrin. Concomitant with this work, I also describe a protein grafting strategy to identify a soluble mimic of S6 ATPase, which is subsequently used to characterize the S6 ATPase/gankyrin interaction by isothermal titration calorimetry.