Spatially Controlled Cell-Free Protein Synthesis and Glycosylation on Chip
Cell Free Systems Conference
2019
Cell Free Systems Conference
Poster Session
Registered Posters
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.