The market for therapeutic proteins is valued near $140 billion annually. Many of these therapeutics are glycoproteins, which require the addition of specific sugars, called 'glycans' at an exact position on the protein through a process called protein glycosylation. The glycan affects protein folding and function and ensures it retains its therapeutic potency. In nature, glycoproteins are produced through a series of sequential reactions inside a cell. Making therapeutic glycoproteins within cells is challenging for a variety of reasons, and extensive and costly purification steps are required to harvest the therapeutic material. With this award, a cell-free glycosylation network will be constructed in a microfluidic device that separates reactions in space and time, giving supreme flexibility in optimizing individual reactions and constructing glycans with high specificity. The benefits of this manufacturing paradigm to society are reducing the cost of these drugs and providing scientists an avenue to design and develop synthetic drug compounds that may or may not exist in nature to treat disease. The related education plan creates a hands-on bio-nanomanufacturing activity for a high school girls organized by the PIs and their student trainees, so that young women will understand the power of biotechnology and be inspired to pursue these career paths.
Cell-free protein synthesis holds great promise for producing high-value, biotherapeutic nanomaterials without cell culture and benefitting from chemical manufacturing know-how. Here, raw materials and biological enzymes are mixed to produce biological products. Shortcomings of this approach are competing reactions, side products, and low yields. Cells avoid these shortcomings by localizing reactions within subcellular compartments and orchestrating the reaction sequences. The biocatalysts that give the final molecule its essential posttranslational features are compartmentalized in membranes. Handling enzymes outside of their native lipid environment can drastically reduce their activity. Thus, in vitro, sequential, bio-enzymatic reactions have never been achieved in a cell-free manner. The research objective is to mimic the elegant compartmentalization strategies used by cells in a microfluidic biomembrane device that organizes biological reactions in proper spatial and temporal sequence. These devices will generate authentically glycosylated proteins. Through assessment of nanostructure product architectures, this work will advance understanding of nanoscale phenomena and processes for nanomaterials manufacture and discovery. This cell-free device concept will enable facile optimization of glycosylated protein production, and provide a framework for understanding how experimental conditions affect product yield and quality that is broadly applicable to the bio-nanomanufacturing of virtually any posttranslationally-modified protein.
