Topological reprogramming transforms an integral membrane oligosaccharyltransferase into a water-soluble glycosylation catalyst

Abstract Glycosyltransferases (GTs) catalyze the formation of new glycosidic bonds and thus are vital for synthesizing nature’s vast repertoire of glycans and glycoconjugates and for engineering glycan-related medicines and materials. However, obtaining detailed structural and functional insights for the >750,000 known GTs is limited by difficulties associated with their efficient recombinant expression. Members of the GT-C fold, in particular, pose the most significant expression challenges due to the integration and folding requirements of their multiple membrane-spanning regions. Here, we address this challenge by engineering water-soluble variants of an archetypal GT-C fold enzyme, namely the oligosaccharyltransferase PglB from Campylobacter jejuni ( Cj PglB), which possesses 13 hydrophobic transmembrane helices. To render Cj PglB water-soluble, we leveraged two advanced protein engineering methods: one that is universal called SIMPLEx ( s olubilization of IMP s with high levels of ex pression) and the other that is custom tailored called WRAPs ( w ater-soluble R Fdiffused a mphipathic p roteins). Each approach was able to transform Cj PglB into a water-soluble enzyme that could be readily expressed in the cytoplasm of Escherichia coli cells at yields in the 3–6 mg/L range. Importantly, solubilization was achieved without the need for detergents and with retention of catalytic function. Collectively, our findings demonstrate that both SIMPLEx and WRAPs are promising platforms for advancing the molecular characterization of even the most structurally complex GTs, while also enabling broader GT-mediated glycosylation capabilities within synthetic glycobiology applications.