Engineered protein hydrogels have emerged as important materials for biomedical applications thanks to their precisely controlled structure and function and their resemblance towards native extracellular matrices. Meanwhile, the design of proteins with nonlinear topologies has arisen as a new branch of protein engineering, yet of which significant applications remain to be seen. This thesis reports the creation of new hydrogel materials using the protein building blocks with uncommon topological architectures.

In Chapter 1, the development and applications of protein topology engineering and protein-based hydrogels are briefly introduced.

In Chapter 2, 3 and 4, we demonstrate the cellular synthesis of a 4-arm star protein, (SpyCatcher)₄GFP, enabled by spontaneous split-GFP reco...[ Read more ]

Engineered protein hydrogels have emerged as important materials for biomedical applications thanks to their precisely controlled structure and function and their resemblance towards native extracellular matrices. Meanwhile, the design of proteins with nonlinear topologies has arisen as a new branch of protein engineering, yet of which significant applications remain to be seen. This thesis reports the creation of new hydrogel materials using the protein building blocks with uncommon topological architectures.

In Chapter 1, the development and applications of protein topology engineering and protein-based hydrogels are briefly introduced.

In Chapter 2, 3 and 4, we demonstrate the cellular synthesis of a 4-arm star protein, (SpyCatcher)₄GFP, enabled by spontaneous split-GFP reconstitution. In combination with the covalent bond-forming SpyTag/SpyCatcher chemistry, this tetra-functional protein serves as a modular building block for creating fluorescent protein networks with varied mechanics, well-suited for cell encapsulation. Conjugating (SpyCatcher)₄GFP with a SpyTagged photoreceptor protein CarH_C leads to another 4-arm star protein, (CarHC)₄GFP, which undergoes rapid solgel and gel-sol transitions in response to AdoB₁₂ and light, respectively. These chemo- and photo-induced phase transitions enable encapsulation and controlled release of protein molecules like fluorescent protein mCherry and biofilm-degrading glycosyl hydrolase PslG, a potential agent for combatting those multidrug-resistant bacterial species such as Pseudomonas aeruginosa involved in chronic infections.

In Chapter 5, we illustrate the creation of a series of dynamically tunable molecular networks through the combined use of SpyTag/SpyCatcher chemistry and physically entangled p53 dimerization domains (Xs). The resulting networks share similar chemical composition but differ significantly in their viscoelasticity. These materials exhibit excellent compatibility towards encapsulated fibroblasts and stem cells. Besides, the impact of relaxation rates on biological processes, like cell morphology developments and bone regenerations are discussed.

Together, this thesis reports the creation of entirely recombinant protein-based hydrogels using uncommon protein architectures, e.g. four-arm star-like molecules and physically interlocked structures, of which the applications such as protein release and cell encapsulation are demonstrated.