THESIS
2020
ix, 58 pages : illustrations (chiefly color) ; 30 cm
Abstract
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...[
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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 AdoB
12-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.
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