Exploiting Chemoenzymatic and Photochemical Reactions to Control 4D Protein Presentation in Cell-Laden Hydrogels

Thesis (Ph.D.)--University of Washington, 2019As the primary workhorses of biology, proteins exhibit a wide range of functions including the ability to regulate cell fate with exquisite specificity. Though proteins represent powerful tools for tissue engineering and drug delivery applications, methods that permit their dynamic and heterogeneous presentation from and within materials remain limited. This thesis exploits recently developed chemoenzymatic strategies to tether biomacromolecules reversibly and site-specifically to hydrogel biomaterials, an approach that uniquely preserves their full bioactivity. First, the versatile Staphylococcus aureus enzyme Sortase A was employed to generate a diverse library of homogenous, singly functionalized fluorescent proteins, enzymes, and growth factors with bioorthogonal reactive handles for biomaterial modification. Additionally, N-myristoyltranferase was used in combination with an engineered photocleavable protein (PhoCl) to create photoreleasable protein chimeras. Photolithographic patterning of these protein constructs was used to pattern dynamic biological response within 3D materials for the first time with subcellular resolution. The techniques presented here enable the creation of next-generation biomaterials that fully utilize the power and versatility of full-length proteins to probe and direct cell function in 4D