Source: Dissertation Abstracts International, Volume: 78-11(E), Section: B.

Adviser: Lynne Regan.

The use of protein nano- and micro-materials as enzyme scaffolds, scaffolds for tissue engineering and drug delivery, and as building blocks for electronic devices requires the controlled self-assembly of these structures into well-defined and programmable geometries and arrays. Here, we present three approaches to protein-based material fabrication centered on the use of modular protein building blocks. First, we present the rational design of two geometric protein arrays. In order to build our first array, we created a toolbox of heterodimeric, orthogonal 8 heptad repeat coiled-coils from concatemers of shorter published coiled-coil sequences. We were also able to build geometric arrays with discrete molecular weights and designed modularity.

In our second approach, we developed two stimuli-responsive, protein-based hydrogel platforms. In the first platform, we designed a hydrogel that forms instantly upon the mixing of two components, and degrades upon treatment with a matrix metalloproteinase (MMP). We determined that this gel system has an elastic modulus of ~ 0.8 kPa, within the range for tissue engineering applications. In the second platform, we developed an ionic hydrogel from the tetratricopeptide repeat (TPR) protein and its cognate C-terminal peptide. By using the SpyTag-SpyCatcher system, we developed a novel method to display the C-terminal peptide, using straightforward recombinant expression.

Finally, we describe a novel approach to create functionalized monolayers using the biofilm surface layer protein A (Bs1A) and the SpyTag-SpyCatcher system. We demonstrate that having the SpyTag peptide fused at the N- or C-terminus of BsIA forms stable monolayers. We establish the creation of stable oil-in-water microcapsules using Bs1A, and also show the fabrication of capsules outwardly displaying the reactive SpyTag peptide. These capsules can be covalently labeled by reacting the surface-displayed SpyTag with SpyCatcher fused to any desired protein. We demonstrate this principle by labeling microcapsules using green fluorescent protein (GFP).