Biology subjects

Sobol, S. E.

Publications and source records attributed to Sobol, S. E..

3 recordsLinked to original sources

bioRxiv2023

Cell-free expression and characterization of multivalent rhamnose-binding lectins using biolayer interferometry

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free protein synthesis as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with BLI analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

synthetic biology↗

bioRxiv2022

A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of travelers diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that significantly reduces mortality and morbidity associated with moderate to severe diarrhea among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic Escherichia coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS confers protection against ETEC infection in mice. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.

synthetic biology↗

bioRxiv2022

A low-cost, thermostable, cell-free protein synthesis platform for on demand production of conjugate vaccines

Cell-free protein synthesis systems that can be lyophilized for long-term, nonrefrigerated storage and transportation have the potential to enable decentralized biomanufacturing. However, increased thermostability and decreased reaction cost are necessary for further technology adoption. Here, we identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability, as well as a lowcost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ~$0.50 per dose after storage at room temperature or 37 {degrees}C for up to 4 weeks and ~$1.00 per dose after storage at 50 {degrees}C for up to 4 weeks. We show that these conjugates generate bactericidal antibodies against enterotoxigenic E. coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our lowcost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.

synthetic biology↗