Biology subjects

Hulbert, S. W.

Publications and source records attributed to Hulbert, S. W..

3 recordsLinked to original sources

bioRxiv2026

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

Glycosyltransferases (GTs) catalyze the formation of new glycosidic bonds and thus are vital for synthesizing natures 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 (CjPglB), which possesses 13 hydrophobic transmembrane helices. To render CjPglB water-soluble, we leveraged two advanced protein engineering methods: one that is universal called SIMPLEx (solubilization of IMPs with high levels of expression) and the other that is custom tailored called WRAPs (water-soluble RFdiffused amphipathic proteins). Each approach was able to transform CjPglB 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.

synthetic biology↗

bioRxiv2025

A scalable cell-free manufacturing platform for two-step bioproduction of immunogenic conjugate vaccines

Rapid and decentralized vaccine production is essential to ensure global preparedness against emerging and re-emerging infectious diseases. Cell-free gene expression systems, which can be freeze-dried for long-term storage and re-activated for point-of-use synthesis, offer a promising solution to address this need. However, scalable cell-free production of conjugate vaccines--highly effective tools against bacterial infections--has been hindered by low yields and inefficient glycosylation. Here, we present an optimized, modular cell-free platform for the synthesis and purification of conjugate vaccines. By decoupling cell-free protein expression from in vitro glycosylation in a two-step approach, we achieve >85% glycosylation efficiency and up to [~]450 mg/L of glycoprotein. We apply this platform to manufacturing protein-polysaccharide conjugates composed of vaccine carrier proteins covalently modified with polysaccharide antigens from enterotoxigenic Escherichia coli O78 and Streptococcus pneumoniae serotype 4. Our workflow produced conjugate vaccine candidates in under 5 days with >87% product purity and low endotoxin levels suitable for preclinical evaluation. Immunization of mice with the pneumococcal conjugate vaccine induced a strong IgG response against the S. pneumoniae serotype 4 capsular polysaccharide, confirming the immunogenicity of the conjugate. We anticipate that this cell-free platform will advance efforts in decentralized manufacturing and rapid response to bacterial disease threats.

synthetic biology↗

bioRxiv2024

Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in Escherichia coli

Human immunoglobulin G (IgG) antibodies are one of the most important classes of biotherapeutic agents and undergo glycosylation at the conserved N297 site in the CH2 domain, which is critical for IgG Fc effector functions and anti-inflammatory activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. While attempts to engineer Escherichia coli for this purpose have been described, they have met limited success due in part to the lack of available oligosaccharyltransferase (OST) enzymes that can install N-linked glycans within the QYNST sequon of the IgG CH2 domain. Here, we identified a previously uncharacterized single-subunit OST (ssOST) from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity and, as a result, was able to catalyze glycosylation of native CH2 domains in the context of both a hinge-Fc fragment and a full-length IgG. Although the attached glycans were bacterial in origin, conversion to a homogeneous, asialo complex-type G2 N-glycan at the QYNST sequon of the E. coli-derived hinge-Fc was achieved via chemoenzymatic glycan remodeling. Importantly, the resulting G2-hinge-Fc exhibited strong binding to human Fc{gamma}RIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of a unique ssOST from D. marinus provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human IgGs and fragments derived thereof.

synthetic biology↗