Browsing by Subject "SpyCatcher"
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Item Open Access Achieving Dynamic Control over Cell Culture Hydrogels Using Engineered Proteins(2020) Hammer, Joshua AIt has been understood for some time that cells are profoundly influenced by their environment. Recently, researchers have made great strides in engineering cell culture platforms that are both physiologically mimetic and reductionist, leading to more biologically relevant cell responses observed within analytically tractable experiments. Moving from stiff tissue culture plastic or glass into hydrogel-based cell culture keeps cells in contact with materials that are stiffness-matched to native tissues, and can reduce or delay de-differentiation into undesirable phenotypes. Incorporating biomolecules like specific adhesion ligands or growth factors into cell culture hydrogels can help drive specific biological outputs, and when coupled with patterning techniques can yield intentional, spatially defined heterogeneity within a cultured cell population. However, biological events or disease states are often are driven by biochemical dynamics, with the time course over which a signal is presented influencing whether cells respond physiologically or pathologically. Unfortunately, dynamic presentations of biomolecules are challenging to replicate within hydrogels due to a lack of ligation mechanisms suitable for dynamically linking relevant biomolecules into the hydrogel matrix in the presence of cells.
In this work, novel protein-based ligation domains were engineered into new strategies for the time-varying presentation of recombinant biomolecules within cell culture hydrogels. SpyCatcher, a protein domain which forms a spontaneous covalent bond with a complementary peptide dubbed “SpyTag”, was used to form a site-specific linkage between a recombinant protein and a synthetic hydrogel. The ligation reaction was shown to proceed under mild conditions appropriate for cell culture, and by adding the cell-adhesive ligand RGDS to the SpyCatcher-tagged protein (forming RGDS-SC), cell spreading within a 3D hydrogel could be switched on by simply adding RGDS-SC topically to cell-laden hydrogels. SnoopCatcher, the chemically orthogonal cousin to SpyCatcher that binds the peptide “SnoopTag”, was then appended with the vascular endothelial growth factor-mimetic peptide QK (KLTWQELYQLKYKGI) to form QK-SnpC, and used in tandem with RGDS-SC to simultaneously control endothelial cell adhesion and mitotic stimulation on synthetic hydrogels containing both Tag peptides.
The Catcher/Tag systems are advantageous due to their specificity and stability. However, their ligations are irreversible because they form covalent bonds. Incorporating the photocleavable fluorophore PhoCl into the backbone of RGDS-SC (forming PhoCl-SC) allowed for the reversible incorporation of a recombinant protein into synthetic hydrogels. SpyCatcher mediated the spontaneous ligation to SpyTag sites within the gel, and by applying 400 nm light, PhoCl was cleaved, thereby removing the N-terminal RGDS tag from the gel. This reversion was limited to one cycle, and so would not be appropriate for presenting a sequence of several biochemical signals, as would be seen by cells near an area of wound healing for instance. To develop a truly reversible conjugation mechanism, the optogenetic protein LOV2 was engineered into a blue light-mediated non-covalent ligation strategy. The LOVTRAP system, consisting of LOV2 and its binding partner Zdk, was shown to allow synthetic gels containing LOV2 to capture Zdk-tagged proteins in the dark, and then release them upon blue light exposure. Because LOV2 will reset to its dark state via thermal relaxation, this capture and release cycle could be repeated at least 3 times, indicating the reversible association of LOVTRAP was functional in a biomaterial setting.
Protein-based ligation domains are simple to use, as they can be added to recombinant proteins genetically, and are inherently site-specific, alleviating worries that protein activity could be compromised upon conjugation to the gel. Moreover, the Catcher systems and LOVTRAP all bind to their binding partners spontaneously under cell culture conditions, reducing the chance that sensitive cell types would be perturbed by their use. These strategies greatly expand the tool kit for dynamically presenting biomolecules to cells via hydrogel immobilization.
Item Open Access Self-Assembled Protein-Based Biomaterials with Tailorable Physical Properties(2015) Goodwin, MorganSoft biomaterials are used in a variety of applications such as scaffolds for cell growth and coatings for implants or transplants. We aim to create a protein hydrogel that will self-assemble upon the mixing of two different protein constructs. This is accomplished using Streptavidin, a protein that tetramerizes, and SpyTag-SpyCatcher, a protein-peptide that spontaneously forms covalent bonds, as the crosslinking mechanisms. Further, using protein building blocks whose viscoelastic properties are known from single-molecule force spectroscopy (SMFS), we aim to create a hydrogel whose physical properties are tailorable by altering the building blocks incorporated in the constructs. This thesis focuses on using an atomic force microscope for force spectroscopy and imaging to analyze the formation of networks upon mixing various protein constructs. We find that small scale networks form using both Streptavidin and SpyTag-SpyCatcher as crosslinkers and that SpyCatcher can be used to detect molecular crosslinking and polyprotein polarization through SMFS.