Protein glycosylation, or the attachment of sugar moieties (glycans) to 
proteins, is important for protein stability, activity, and immunogenicity. 
However, understanding the roles and regulations of site-specific glycosylation 
events remains a significant challenge due to several technological limitations. 
These limitations include a lack of available tools for biochemical 
characterization of enzymes involved in glycosylation. A particular challenge is 
the synthesis of oligosaccharyltransferases (OSTs), which catalyze the 
attachment of glycans to specific amino acid residues in target proteins. The 
difficulty arises from the fact that canonical OSTs are large (>70 kDa) and 
possess multiple transmembrane helices, making them difficult to overexpress in 
living cells. Here, we address this challenge by establishing a bacterial 
cell-free protein synthesis platform that enables rapid production of a variety 
of OSTs in their active conformations. Specifically, by using lipid nanodiscs as 
cellular membrane mimics, we obtained yields of up to 420 μg/ml for the 
single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter 
jejuni, as well as for three additional PglB homologs from Campylobacter coli, 
Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes 
catalyzed N-glycosylation reactions in vitro with no purification or processing 
needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to 
glycosylate multiple target proteins with varying N-glycosylation acceptor 
sequons. We anticipate that this broadly applicable production method will 
advance glycoengineering efforts by enabling preparative expression of 
membrane-embedded OSTs from all kingdoms of life.