(158l) Spatially Controlled Cell-Free Protein Synthesis and Glycosylation on Chip

Among cellular processes, glycosylation of biomolecules like proteins or lipids is critical for their proper function and efficacy. Recombinant protein production is a highly conserved, templated process, while added glycans are extremely heterogenous and proper control over their expression is important in controlling biological activity and therapeutic function. Current cell-based methods to produce glycosylated biologics rely on native machinery, which inherently produces non-specific glycan profiles, making purification difficult as well as time and labor intensive. To solve these issues, cell-free technologies have been developed to produce proteins without growth constraints and allow the addition of cell-free glycosylation pathways.

I will describe our work towards a microfluidic platform analogous to the natural eukaryotic cellular assembly line, that integrates spatially separated cell-free protein synthesis, glycosylation, and enrichment of a model glycoprotein. Microfluidics allow for tight control over environmental conditions, recyclability of tethered enzymes, and importantly, separation of reactions that has yet to have been achieved in cell-free glycosylation systems. As an initial proof-of-concept, the green fluorescent protein (GFP) is used to allow easy visualization of cell-free protein production and purification. In the first module, GFP is expressed with continuous flow using E. coli cell-free extract. In the second module, the GFP with a terminal glycosylation tag is passed through a microfluidic chamber with glycosylation machinery. The current system uses the integral membrane oligosaccharyltransferase PglB, which efficiently transfers a heptasaccharide glycan from its lipid donor to the protein. For continuous production of glycoprotein, PglB is biotinylated and immobilized using the biotin-streptavidin interaction. In the third module, the GFP product is captured using immobilized affinity chromatography to allow for easy capture and subsequent release of the His-tag protein. This work presents a novel solution to cell-free systems that enables fundamental studies of glycosylation as well as being used for continuous cell-free glycoprotein production.