Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer 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 expression 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 bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the 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.

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 traveler's 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 appreciably decreases mortality and morbidity associated with ETEC infection 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 E. 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 reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. 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.

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, non-refrigerated 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 and a low-cost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ∼$0.50 per dose after storage at room temperature (∼22 °C) or 37 °C for up to 4 weeks, and ∼$1.00 per dose after storage at 50 °C for up to 4 weeks, with costs based on raw materials purchased at the laboratory scale. We show that these conjugate vaccines generate bactericidal antibodies against enterotoxigenic Escherichia coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our low-cost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.