An intermediate step in the synthesis of most pharmaceutical products, including therapeutic proteins, genome-enabled personalized medicine, and anti-viral vaccines, is the isolation of the target compound of interest from a complex biological mixture. Separation of the species of interest is a complex, resource-intensive multi-step process, but is essential to ensure the purity and quality of the resulting pharmaceutical product. Pharmaceuticals are generally separated via chromatography, in which a complex mixture is passed over multiple beds packed with solid resin particles. Although the solids are generally inert, different components of the mixture pass through the bed at different velocities, as dictated by the size of each molecule present in the mixture, its travel path, and interactions with the solid surface that may impede motion. This project will develop new chromatographic resins that more efficiently separate biological mixtures, by imparting chemical ligands to the solid that have binding interactions with the compounds of interest that selectively impede their motion through the packed column. It is anticipated that such multimodal ligands, i.e. those that have multiple interactions with specific compounds, will significantly reduce the complexity, and thus the cost, of chromatographic purification of biological mixtures. Specifically, this project will genetically engineer the active sites of enzymes, such that the enzymes will synthesize designer ligands with properties that make them more selectively bind to targeted proteins from the complex mixture. The binding sites to be developed in this project are therapeutic protein-specific glycan-based ligands (or glycoligands), which will be synthesized via a novel biosynthetic approach fusing engineered glycosynthase enzymes. These enzymes offer several advantages over traditional approaches, including that they can be easily engineered to tailor the reaction specificity to produce highly biocompatible ligands for protein purification. The central hypothesis of this work is that genetic engineering of multifunctional glycosynthase enzymes will lead to the ability of these enzymes to finely tailor glycoligands, which in turn, will have desirable selectivity for multimodal ligand based protein chromatography. A library of enzymes will be designed to produce a library of active and selective binding ligand resins. The resulting multimodal interactions, including recognition, binding, and unbinding, will be characterized using a high throughput experimental platform. Glycoligands synthesized using various glycone and aglycone moieties with fine-tuned hydrogen bonding, hydrophobic, and/or electrostatic interactions will facilitate targeted purification of proteins with defined surface properties. This work will lead to the development of new biomaterials, experimental tools, and quantitative structure-property relationship models for predicting protein-glycan interactions that will advance diverse fields ranging from glycobiology to bioseparations. Undergraduate and graduate students will be trained in the interdisciplinary areas of protein engineering and bioseparations, and concepts from the research will be incorporated into an undergraduate course and into an outreach/training program at Rutgers University. The PI will partner with university and industrial partners to test the commercial potential of the designer glycoligands.
|Effective start/end date||8/15/17 → 7/31/20|
- National Science Foundation (NSF)