Assembling engineered protein molecules into bioactive materials offers great promise for biomedical applications including drug delivery and tissue regeneration. Rapid developments have been made in the field of protein assembly over the past few decades. It has been widely recognized that entirely protein-based materials arising from protein assembly often possess some important merits such as superior biocompatibility, genetic programmability and amenability toward diverse biofunctionalization. However, protein materials may suffer from some drawbacks such as the limitations in mechanical tunability. Here we investigated several protein assembly strategies for designing protein materials.

Firstly, inspired by naturally occurring mussel foot proteins, we demonstrate the synt...[ Read more ]

Assembling engineered protein molecules into bioactive materials offers great promise for biomedical applications including drug delivery and tissue regeneration. Rapid developments have been made in the field of protein assembly over the past few decades. It has been widely recognized that entirely protein-based materials arising from protein assembly often possess some important merits such as superior biocompatibility, genetic programmability and amenability toward diverse biofunctionalization. However, protein materials may suffer from some drawbacks such as the limitations in mechanical tunability. Here we investigated several protein assembly strategies for designing protein materials.

Firstly, inspired by naturally occurring mussel foot proteins, we demonstrate the synthesis of protein hydrogels by photochemically crosslinking recombinant mussel foot protein-3 (Mfp3) heterologously produced by Escherichia coli. The hydrogels’ stiffness can be tuned by adjusting the concentration of protein polymers or co-oxidants needed for the photochemistry. The protein polymers were also designed to contain RGD cell-binding ligands and SpyCatcher domains, the latter of which enabled post-gelation decoration with diverse folded globular proteins under mild physiological conditions. Not only did the resulting hydrogels support the adhesion and proliferation of a variety of cell lines, but also were able to activate JAK/STAT3 pathway and induce neurite growth via covalently immobilized leukemia inhibitory factor (LIF). These results illustrate a new strategy for designing bioactive materials for regenerative neurobiology. Secondly, we present a simple strategy for creating injectable, photoresponsive protein hydrogels through metal-directed assembly of His6-tagged photoreceptor protein CarHc. Addition of cofactor AdoB12 into metal/protein complexes leads to rapidly gelation, which exhibit self-healing properties. Decorating this injectable hydrogel with leukemia inhibitory factor (LIF) via Zn²⁺ coordination further led to prolonged STAT3 activation and enhanced axon regeneration in mouse optic nerves. The resulting materials illustrates a general approach for designing injectable protein materials with widely potential application. Finally, we examined the feasibility of creating protein hydrogels suitable for 3D culturing of neurons through the combined use of an ultra-strong protein-protein interaction pair, IM7/CL7, and SpyTag/SpyCatcher chemistry. The use of SpyTag/SpyCatcher chemistry enabled the creation of building blocks with uncommon topologies, such as 4-arm star proteins, of which the supramolecular assembly via IM7/CL7 further led to the formation of protein hydrogels. The resulting hydrogels with self-healing abilities could serve as a promising alternative for neuronal culture and axon regeneration.

In summary, this thesis illustrates the new strategies for protein assembly that has opened a door to diverse advanced biomaterials. We anticipate that these studies will provide a new way of thinking for protein engineering and materials science.

Key words: Protein assembly, hydrogel, protein topology engineering, SpyTag/SpyCatcher, mussel foot protein