Protein Engineering for Biomedicine and Beyond
McCord, Jennifer Phipps
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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.
General Audience Abstract
Many applications in medicine and research require molecular sensors that bind their target tightly and selectively, even in complex mixtures. Mammalian antibodies are the best-studied examples of these sensors, but problems with the stability, expense, and selectivity of these antibodies have led to the development of alternatives. In the search for better sensors, repeat proteins have emerged as one promising class, as repeat proteins are relatively simple to design while being able to bind specifically and selectively to their targets. However, a drawback of commonly used designed repeat proteins is that their targets are typically restricted to proteins, while many targets of biomedical interest are sugars, such as those that are responsible for blood types. Repeat proteins from the immune system, on the other hand, bind targets of many different types. We looked at the unusual immune system of a freshwater polyp as inspiration to design a new repeat protein to recognize nonprotein targets. My second research project focused on binding cellulose, as it is the most abundant and inexpensive source of biological matter and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept cellulose-based fuels from competing commercially with biofuel made from corn and other starchy plants. One strategy to lower costs relies on using helper proteins to reduce the amount of enzyme needed to break down the cellulose, as enzymes are the most expensive part of processing. We designed such a protein for this function to be more stable than natural proteins currently used. The resulting designed protein binds to multiple cellulose structures. Designing a protein from scratch also allows us to study small changes to the binding site, allowing us to better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal for tissue engineering and wound healing applications. However, the process by which these proteins are extracted from hair leads to some protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Making these proteins synthetically allows us to have pure starting material, and lets us add new features to the proteins, which translates into materials better tailored for their applications. We discuss here one example, in which we added a cell-binding motif to a keratin protein sequence.
- Doctoral Dissertations