Spatially Controlled Cell-Free Protein Synthesis and Glycosylation on Chip

Among cellular processes, glycosylation of biomolecules such as proteins or lipids is critical for their proper function and efficacy. Recombinant protein production is a highly conserved and templated process, while post-translational modifications, like glycans, rely on kinetic availability of enzymes which allows for nearly infinite configurations. This makes proper control over their production important in directing biological activity and therapeutic function. Current cell-based methods to produce glycosylated biologics inherently give heterogenous 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 designer 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. Microfluidic systems allow for tight control over environmental conditions, recyclability of tethered enzymes, and importantly, separation of reactions that has yet to be 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 synthesis, glycosylation, 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 though a microfluidic chamber with glycosylation machinery where an oligosaccharyltransferase, PglB, efficiently transfers a heptasaccharide glycan from its lipid donor to the protein. In the third module, the glycosylated GFP product is captured using a Ni-NTA surface to allow for easy concentration and subsequent release of the hexahistidine-tagged protein. This work presents a novel solution to cell-free systems that enables fundamental studies of the glycosylation mechanism and opens the door to continuous cell-free glycoprotein production.