In vitro glycosylation by cell-free protein synthesis-derived oligosaccharyltransferases

Asparagine (N)-linked glycosylation is an important protein modification that is ubiquitous in nature and therapeutically relevant.  The crucial cellular and molecular processes affected by N-glycosylation are far reaching, including protein folding, immunogenicity, activity, half-life, and regulation of signaling cascades. Despite its vast importance, our ability to study functional and structural consequences of site-specific glycosylation remains limited. For these reasons, developing tools to study individual glycosylation components expands our ability to understand and engineer glycosylation for developing novel therapeutics.  One such component is the large, membrane-bound enzyme, termed an oligosaccharyltransferase (OST).  The OST is responsible for catalyzing the formation of glycosidic bonds between a target protein bearing an asparagine residue within a consensus sequence and lipid-linked oligosaccharides.  Understanding and studying OSTs is extremely important since they dictate where a glycan is attached, and which olicosaccharide is attached.  It has been shown elsewhere that full heterologous glycosylation pathways can be expressed in E. coli, conferring the strain with the ability to glycosylate protein. This method, however, does not allow for user-defined tuning of glycosylation components, and relies heavily on the viability of cells. In particular, the over-expression of membrane proteins- such as OSTs- can be highly toxic to cells and impede expression.  In order to address these issues, we have leveraged the E. coli cell-free protein synthesis (CFPS) platform to rapidly express and study six bacterial OSTs.  By optimizing CFPS for use with lipid-protein nanodiscs, we are able to synthesize properly-folded OSTs whose transmembrane stretches are supported within the lipid bilayer of the nanodiscs.  This method results in up to 750 ug/mL of soluble enzyme in six hours.  We show that CFPS-derived OSTs can then be directly introduced into in vitro glycosylation (IVG) reactions without the need for lengthy purifications or overexpression.  These CFPS-derived OSTs are active on multiple protein substrates, and site-specifically glycosylate target proteins comparably to in vivo produced OSTs. In addition, the chemical environments of IVG reaction conditions can be easily optimized for higher glycosylation efficiencies, and can be tuned to any OST.  By simply optimizing buffer concentration, supplementation of cofactors, and addition of macromolecular crowding reagents, we have improved efficiency of IVG reactions by over 50%. We anticipate this broadly applicable method for quickly manufacturing and studying OSTs will provide new opportunities for studying glycosylation components, thereby bringing us closer to applying this knowledge to develop novel glycoprotein therapeutics.