Protein Engineering for Biomedicine and Beyond

Abstract

Many applications in biomedicine, research, and industry require recognition agents with specificity and selectivity for their target. Protein engineering enables the design of scaffolds that can bind targets of interest while increasing their stability, and expanding the scope of applications in which these scaffolds will be useful. Repeat proteins are instrumental in a wide variety of biological processes, including the recognition of pathogen-associated molecular patterns by the immune system. A number of successes using alternative immune system repeat protein scaffolds have expanded the scope of recognition agents available for targeting glycans and glycoproteins in particular. We have analyzed the innate immune genes of a freshwater polyp and found that they contained particularly long contiguous domains with high sequence similarity between repeats in these domains. We undertook statistical design to create a binding protein based on the H. magnipapillata innate immune TPR proteins. My second research project focused on creating a protein to bind cellulose, as it is the most abundant and inexpensive source of biomass and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept lignocellulosic fuels from competing commercially with starch-based biofuels. In recent years a strategy to protect processing enzymes with synergistic proteins emerged to reduce the amount of enzyme necessary for lignocellulosic biofuel production. Simultaneously, protein engineering approaches have been developed to optimize proteins for function and stability enabling the use of proteins under non-native conditions and the unique conditions required for any necessary application. We designed a consensus protein based on the carbohydrate-binding protein domain CBM1 that will bind to cellulosic materials. The resulting designed protein is a stable monomeric protein that binds to both microcrystalline cellulose and amorphous regenerated cellulose thin films. By studying small changes to the binding site, we can better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications such as enhancing the saccharification of lignocellulosic feedstock for biofuel production. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal scaffolds for tissue engineering and wound healing. However, the extraction process leads to protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Recombinant keratin biomaterials are free from these disadvantages, while heterologous expression of these proteins allows us to manipulate the primary sequence. We endeavored to add an RGD sequence to facilitate cell adhesion to the recombinant keratin proteins, to demonstrate an example of useful sequence modification.