Hydrogel is a widely studied biomaterial candidate due to its high biocompatibility. In recent years, thanks to the well-established strategy of designing and producing recombinant protein, peptide/protein pairs, such as SpyTag/SpyCatcher and SnoopTag/SnoopCatcher, which exhibit robust covalent bonding behavior comparable to synthetic "click" chemistry, have been created and further adopted for developing various biomedical tools. These powerful protein chemistries can be regarded as genetically encoded click chemistry (GECC). The prototypic GECC, SpyTag/SpyCatcher, has enabled the creation of various entirely protein-based hydrogels with different functionalities, as well as the conceptualization of protein topology engineering—an emerging methodology with emphasis on the preci...[ Read more ]

Hydrogel is a widely studied biomaterial candidate due to its high biocompatibility. In recent years, thanks to the well-established strategy of designing and producing recombinant protein, peptide/protein pairs, such as SpyTag/SpyCatcher and SnoopTag/SnoopCatcher, which exhibit robust covalent bonding behavior comparable to synthetic "click" chemistry, have been created and further adopted for developing various biomedical tools. These powerful protein chemistries can be regarded as genetically encoded click chemistry (GECC). The prototypic GECC, SpyTag/SpyCatcher, has enabled the creation of various entirely protein-based hydrogels with different functionalities, as well as the conceptualization of protein topology engineering—an emerging methodology with emphasis on the precise synthesis of nonlinear biomacromolecules using engineered cellular machinery. Cellular synthesis of 4-arm star-like proteins have recently been demonstrated via the co-expression of self-assembling split-GFP fragments—noncovalent though—inside bacterial cells, of which controlled assembly further led to the creation of a variety of stimuli-responsive protein materials with well-defined structures. Nevertheless, the cellular synthesis of genuine 4-arm star protein molecules (covalent), as well as its use in material design, has yet to be achieved.

In this thesis, we leveraged another GECC pair, SnoopTag/SnoopCatcher, which is orthogonal to SpyTag/SpyCatcher, and successfully created 4-arm star proteins, (SpyCatcher)₄Snoop, through cellular synthesis. Using this uncommon protein building block and SpyTag/SpyCatcher chemistry, we further synthesized several protein networks comprising stimuli-responsive domains, including sequence-specific nickel-assisted cleavage tag (SNAC) and AdoB₁₂-dependent photoreceptor protein CarH_C. The resulting protein hydrogels have been marked by their robust stimuli-responsiveness and cytocompatibility. This study illustrates protein topology engineering enabled by GECC as a new strategy for designing smart protein materials.