On July 8, 2025, Bidstrup et al., representing institutions such as Cornell University, the University of Maryland, and Stanford University, published a paper entitled "Glycosylation of Structured Protein Domains in Cell-Free Reaction Environments" in ACS Synthetic Biology. The article addresses a fundamental limitation in biomanufacturing: the challenge of predictably attaching N-linked glycans to protein targets with constrained acceptor sites. The main finding is the successful, efficient glycosylation of proteins with structured domains—like ribonuclease A (RNase A) and the fragment crystallizable (Fc) region of immunoglobulin G (IgG)—using an E. coli-based Cell-Free Glycoprotein Synthesis (CFGpS) System. The authors propose a unique co-translational mechanism that circumvents the structural barriers that foil traditional in vitro methods.

Overview

The drive for this research stems from the pressing need for decentralized and customizable biomanufacturing of complex protein therapeutics and vaccines. Most clinically approved protein therapeutics are glycoproteins, and the attached glycans are vital for efficacy, stability, and immunogenicity. Traditional manufacturing in Mammalian Cell Lines is costly, time-consuming, and difficult to customize. The field of synthetic biology offers solutions through highly controllable platforms.

The CFGpS platform emerged as a promising solution. By utilizing extracts from glycocompetent bacteria (engineered to possess the necessary machinery like oligosaccharyltransferase (OST) and lipid-linked oligosaccharides (LLOs)) and supplementing them with DNA, it enables the rapid, single-pot biosynthesis of glycoproteins. While CFGpS offers modularity and speed, a key limitation has been whether it could replicate the complex, temporally controlled glycosylation of highly structured proteins that occurs in living cells, where protein folding and membrane transport are closely coordinated. This new research directly addresses that mechanistic gap, proving that the cell-free environment inherently possesses a unique mechanism to overcome structural constraints.

Research Results

• Challenging Structured Domains as Acceptor Targets

The researchers selected two key eukaryotic glycoproteins: bovine RNase A and the Fc region of human IgG. These proteins are formidable targets because their natural glycan acceptor sites (sequons) are situated in highly structured, constrained regions. Previous studies demonstrated that once these proteins are fully folded, the bacterial OST, PglB, struggles to access the acceptor sites. This established preference for flexible, solvent-exposed motifs meant that efficient glycosylation of these targets was considered possible only during or immediately after membrane translocation (in vivo).

• Efficient Glycosylation Under Cell-Free Conditions

Using the CFGpS system—which integrates transcription, translation, and glycosylation into a single-pot extract—the team observed efficient glycosylation of both the structured RNase A and the Fc region. This result was a critical surprise, as the proteins were being synthesized in an extract, not a living cell, yet the efficiency far surpassed that of traditional in vitro glycosylation (IVG). In IVG experiments, where already-folded, purified proteins were incubated with the OST and LLOs, glycosylation was minimal, confirming the sequons are indeed inaccessible post-folding. The CFGpS system clearly provides a temporary window of opportunity for the OST to engage the buried sites.

Fig.1 Glycosylation mechanisms in bacterial cell-based and cell-free expression systems. (Bidstrup, et al., 2025)

Fig.2 Efficient glycosylation of the structured domain in RNase A using CFGpS. (Bidstrup, et al., 2025)

• Novel Co-Translational Mechanism Discovered

To unravel this efficiency paradox, the team investigated the role of protein synthesis and membrane components. Crucially, they demonstrated that efficient glycosylation was dependent on active ribosomal translation. Glycosylation dropped precipitously when translation was inhibited. However, unlike in vivo glycosylation—which often requires a signal peptide to guide the protein to the membrane for translocation—the cell-free process did not require signal peptide-mediated translocation. This led to the proposal of a novel co-translational, but not co-translocational, glycosylation mechanism. In this model, the nascent polypeptide chain is partially modified by the OST while it is still being synthesized by the ribosome, before the structured domain has fully folded into its restrictive conformation. This transient, flexible state is the key to unlocking these difficult acceptor sites.

Conclusion

By demonstrating that the CFGpS platform efficiently glycosylates acceptor sites within structurally constrained protein domains—targets previously considered difficult or impossible to modify outside of complex cellular environments—the research significantly expands the practical utility of cell-free biomanufacturing. The newly proposed co-translational glycosylation mechanism provides a fundamental mechanistic understanding, confirming that the cell-free reaction environment itself offers a transient period of structural plasticity ideal for enzyme-substrate engagement. This innovation moves CFGpS closer to becoming a truly viable, high-throughput platform for the rapid, low-cost, and customizable production of complex eukaryotic glycoprotein targets, including next-generation glycoconjugate vaccines and sophisticated therapeutic antibodies.