We use a droplet-microfluidics-based platform to rapidly identify and isolate individual primary cells that secrete desired antibodies. We then retrieve the antibody-encoding sequences and create recombinant antibodies that bind the target protein.

Dendritic cell (DC)-based vaccines have shown promise as an immunotherapeutic modality for cancer and infectious diseases in many preclinical studies and clinical trials. Provenge (sipuleucel-T), a DC-based vaccine based on ex vivo-generated autologous DCs loaded with antigens, has recently received FDA approval for prostate cancer treatment, further validating the potential of DC-based vaccine modalities. However, direct antigen delivery to DCs in vivo via DC-specific surface receptors would enable a more direct and less laborious approach to immunization. In this study, the recombinant extracellular domains (ECD) of human and mouse DC-SIGN (hDC-SIGN and mDC-SIGN) were generated as DC-specific targets for mRNA display. Accordingly, an antibody-mimetic library was constructed by randomizing two exposed binding loops of an expression-enhanced 10th human fibronectin type III domain (e10Fn3). After three rounds of selection against mDC-SIGN, followed by four rounds of selection against hDC-SIGN, we were able to evolve several dual-specific ligands, which could bind to both soluble ECD of human and mouse DC-SIGNs. Using a cell-binding assay, one ligand, eFn-DC6, was found to have high affinity to hDC-SIGN and moderate affinity to mDC-SIGN. When fused with an antigenic peptide, eFn-DC6 could direct the antigen delivery and presentation by human peripheral blood mononuclear cell (PBMC)-derived DCs and stimulate antigen-specific CD8(+) T cells to secrete inflammatory cytokines. Taken together, these results demonstrate the utility of mRNA display to select protein carriers for DC-based vaccination and offer in vitro evidence that the antibody-mimetic ligand eFn-DC6 represents a promising candidate for the development of an in vivo DC-based vaccine in humans.

Single-span membrane proteins (ssMPs) represent approximately one-half of all membrane proteins and play important roles in cellular communications. However, like all membrane proteins, ssMPs are prone to misfolding and aggregation because of the hydrophobicity of transmembrane helices, making them difficult to study using common aqueous solution-based approaches. Detergents and membrane mimetics can solubilize membrane proteins but do not always result in proper folding and functionality. Here, we use cell-free protein synthesis in the presence of oil drops to create a one-pot system for the synthesis, assembly, and display of functional ssMPs. Our studies suggest that oil drops prevent aggregation of some in vitro-synthesized ssMPs by allowing these ssMPs to localize on oil surfaces. We speculate that oil drops may provide a hydrophobic interior for cotranslational insertion of the transmembrane helices and a fluidic surface for proper assembly and display of the ectodomains. These functionalized oil drop surfaces could mimic cell surfaces and allow ssMPs to interact with cell surface receptors under an environment closest to cell-cell communication. Using this approach, we showed that apoptosis-inducing human transmembrane proteins, FasL and TRAIL, synthesized and displayed on oil drops induce apoptosis of cultured tumor cells. In addition, we take advantage of hydrophobic interactions of transmembrane helices to manipulate the assembly of ssMPs and create artificial clusters on oil drop surfaces. Thus, by coupling protein synthesis with self-assembly at the water-oil interface, we create a platform that can use recombinant ssMPs to communicate with cells.embrane proteins populate the surfaces of cells and allow cells to sense and interact with their external environments. Membrane proteins constitute 25-30% of all proteins identified in sequenced genomes, and understanding their functions is important, because they represent the majority of current drug targets (1). Conventionally, function is inferred from structural studies of membrane proteins; these studies involve expression and purification of natural or recombinant membrane proteins followed by crystallization. However, membrane proteins are notoriously hard to work with because of their hydrophobic domains that can cause misfolding and aggregation (2). Consequently, detergents and membrane mimetics (3) are used to solubilize membrane proteins. To avoid cytotoxicity caused by in vivo expression and enable highthroughput production, cell-free systems are used to express membrane proteins in vitro (4-7). Despite recent progresses, understanding the functions of membrane proteins is still a lengthy and difficult process.Here, we develop a one-pot approach for synthesis, assembly, and display of single-span membrane proteins (ssMPs) for direct functional studies. ssMPs contain a single transmembrane (TM) helix that anchors the hydrophilic ectodomains on the membrane surface; ssMPs represent approximately one-half of all membrane proteins and are involved ...

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Chlamydia use a complex of outer envelope proteins, which are highly cross-linked by disulfide bonds, to protect their infectious developmental form from lysis. Reported herein are the NMR chemical shift assignments of DsbH, a novel disulfide oxidoreductase from Chlamydia.