Exploration of microfluidic technologies and eukaryotic oligosaccharyltransferases to improve Escherichia coli-based glycoprotein production
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A key feature in manufacturing biologics is glycosylation, a posttranslational modification where a glycan is attached to amino acid side chains of proteins. Glycosylation gives rise to diverse chemical structures that affect drug properties such as stability, activity, and immunogenicity. Traditionally, glycoproteins are produced in eukaryotic cells where glycosylation is not template based, giving rise to heterogeneous mixtures of glycoforms that are difficult to parse out. In response to this challenge, cell-free technologies have emerged that enable production of glycoproteins outside of the cell. In my dissertation work, I address two bottlenecks of cell-free production of glycoproteins. First, current cell-free technologies use a “one-pot” system where integration of complex reaction pathways can lead to competing reactions and generation of side products. In my work, I have integrated cell-free technologies into a microfluidic system where individual bioprocesses are compartmentalized to enable greater control and optimization at each step. Second, to expand the range of human-like glycoproteins that can be produced, I examine single subunit OSTs from eukaryotic sources for use in E. coli that I believe will be amenable to cell-free systems. Together, these advancements bring us closer to the ability to perform bottom-up construction of designer, structurally uniform glycoproteins for study and use as therapeutics.
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Wu, Mingming