Many therapeutic proteins are decorated with complex carbohydrates, called glycans, that are important for recognition and function, and the lack of a robust method for adding those glycans has been a stumbling block to wider adoption of cell-free biomanufacturing for making therapeutics and vaccines on demand. A team led by Michael C. Jewett of Northwestern University and Matthew P. DeLisa of Cornell University reports a single-pot method for synthesizing glycoproteins (Nat. Commun. 2018, DOI: 10.1038/s41467-018-05110-x). To Escherichia coli, which doesn’t make glycoproteins naturally, the researchers added plasmids from glycoprotein-synthesizing bacteria: one encoding enzymes that make desired glycans and another encoding machinery that attaches the glycans to the target protein. They then lysed the E. coli cells, removed cell membranes, and used the resulting extract for synthesizing various proteins with glycans added site-selectively to asparagine residues. They were able to incorporate glycans with a trimannose core structure, which is a first step to being able to add glycans with branching patterns akin to those on therapeutic antibodies. They were also able to add sugars to all three glycosylation sites in the protein erythropoietin. In future work, the researchers plan to extend the cell-free approach to mammalian glycans and glycosylation patterns.