Figure 4.

Glycoprotein production in cell-free systems. The Campylobacter jejuni N-glycan biosynthesis pathway, which contains glycosyltransferases and a flippase, is expressed in vivo in Escherichia coli. These enzymes assemble sugar monomers (bacillosamine, N-acetylglucosamine, glucose) onto a lipid anchor (undecaprenyl pyrophosphate) in the membrane to form lipid-linked oligosaccharides (LLOs) (top). Similarly, the C. jejuni oligosaccharyltransferase (OST), PglB, is expressed in vivo in E. coli (bottom). LLOs and PglB are purified and added to the in vitro glycoprotein synthesis reaction. Addition of purified LLOs and PglB to the cell-free protein synthesis (CFPS) reaction results in the synthesis of fully glycosylated AcrA, a C. jejuni glycoprotein (Guarino and DeLisa 2012).