RESUMEN
Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells. We used a defined mixture of hybridoma cells to characterize the system, sorting droplets at up to 100 Hz and isolating desired hybridoma cells, comprising 0.1% of the input, with a false positive rate of less than 1%. We then applied the system to once-frozen primary B-cells to isolate rare cells secreting target-binding antibody. We performed RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences, and we used rapid cell-free protein synthesis to generate single-chain variable fragment-format (scFv) antibodies from fourteen of the sorted cells. Twelve of these showed antigen-specific binding by ELISA. Our platform facilitates screening animal B-cell repertoires within days at low cost, increasing both rate and range of discovering antigen-specific antibodies from living organisms. Further, these techniques can be adapted to isolate cells based on virtually any secreted product.
RESUMEN
Bacteria in the environment play a major role in the degradation of widely used man-made recalcitrant organic compounds. Pseudomonas nitroreducens TX1 is of special interest because of its high efficiency to remove nonionic ethoxylated surfactants. In this study, a novel approach was demonstrated by a bacterial enzyme involved in the formation of radicals to attack ethoxylated surfactants. The dihydrolipoamide dehydrogenase was purified from the crude extract of strain TX1 by using octylphenol polyethoxylate (OPEOn) as substrate. The extent of removal of OPEOs during the degradation process was conducted by purified recombinant enzyme from E. coli BL21 (DE3) in the presence of the excess of metal mixtures (Mn2+, Mg2+, Zn2+, and Cu2+). The metabolites and the degradation rates were analyzed and determined by liquid chromatography-mass spectrometry. The enzyme was demonstrated to form Fenton reagent in the presence of an excess of metals. Under this in vitro condition, it was shown to be able to shorten the ethoxylate chains of OPEOn. After 2 hours of reaction, the products obtained from the degradation experiment revealed a prominent ion peak at m/z = 493.3, namely the ethoxylate chain unit is 6 (OPEO6) compared to OPEO9 (m/z = 625.3), the main undegraded surfactant in the no enzyme control. It revealed that the concentration of OPEO15 and OPEO9 decreased by 90% and 40% after 4 hours, respectively. The disappearance rates for the OPEOn homologs correlated to the length of the exothylate chains, suggesting it is not a specific enzymatic reaction which cleaves one unit by unit from the end of the ethoxylate chain. The results indicate the diverse and novel strategy by bacteria to catabolize organic compounds by using existing housekeeping enzyme(s).
Asunto(s)
Proteínas Bacterianas/metabolismo , Dihidrolipoamida Deshidrogenasa/metabolismo , Fenoles/química , Fenoles/metabolismo , Pseudomonas/enzimología , Tensoactivos/química , Tensoactivos/metabolismo , Cromatografía Liquida , Cobre/metabolismo , Escherichia coli/enzimología , Magnesio/metabolismo , Manganeso/metabolismo , Espectrometría de Masas , Zinc/metabolismoRESUMEN
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.
Asunto(s)
Bioquímica/métodos , Proteínas de la Membrana/síntesis química , Aceites/química , Agua/química , Apoptosis , Electroforesis en Gel de Poliacrilamida , Proteína Ligando Fas/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Jurkat , Ligando Inductor de Apoptosis Relacionado con TNF/síntesis químicaRESUMEN
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.
Asunto(s)
Antígenos/administración & dosificación , Moléculas de Adhesión Celular/genética , Técnicas de Visualización de Superficie Celular/métodos , Células Dendríticas/inmunología , Sistemas de Liberación de Medicamentos/métodos , Lectinas Tipo C/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Animales , Anticuerpos/inmunología , Antígenos/genética , Secuencia de Bases , Sitios de Unión , Materiales Biomiméticos , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Ligandos , Ratones , Datos de Secuencia Molecular , ARN Mensajero/inmunología , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The Chlamydia family of human pathogens uses outer envelope proteins that are highly cross-linked by disulfide bonds but nevertheless keeps an unusually high number of unpaired cysteines in its secreted proteins. To gain insight into chlamydial disulfide bond catalysis, the structure, function, and substrate interaction of a novel periplasmic oxidoreductase, termed DsbH, were determined. The structure of DsbH, its redox potential of -269 mV, and its functional properties are similar to thioredoxin and the C-terminal domain of DsbD, i.e. characteristic of a disulfide reductase. As compared with these proteins, the two central residues of the DsbH catalytic motif (CMWC) shield the catalytic disulfide bond and are selectively perturbed by a peptide ligand. This shows that these oxidoreductase family characteristic residues are not only important in determining the redox potential of the catalytic disulfide bond but also in influencing substrate interactions. For DsbH, three functional roles are conceivable; that is, reducing intermolecular disulfides between proteins and small molecules, keeping a specific subset of exported proteins reduced, or maintaining the periplasm of Chlamydia in a generally reducing state.
Asunto(s)
Chlamydia/enzimología , Cisteína/metabolismo , Disulfuros/metabolismo , Proteínas Fúngicas/metabolismo , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Chlamydia/genética , Chlamydia/patogenicidad , Infecciones por Chlamydia/fisiopatología , Secuencia Conservada , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Modelos Moleculares , Biología Molecular , Datos de Secuencia Molecular , Oxidorreductasas/química , Oxidorreductasas/genética , Conformación Proteica , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Chlamydophila pneumoniae/enzimología , Disulfuros/química , Proteínas Fúngicas/química , Espectroscopía de Resonancia Magnética/métodos , Oxidorreductasas/química , Secuencia de Aminoácidos , Isótopos de Carbono/química , Peso Molecular , Isótopos de Nitrógeno/química , Estructura Terciaria de Proteína , ProtonesRESUMEN
A novel octahedral complex CoII(HAPP)(TFA)2 [hexaazaphenantholine-cyclophane (HAPP), trifluoroacetate (TFA)] is a DNA bulge-specific probe with single-strand DNA cleavage activity in the presence of H2O2. This complex exhibits low affinity towards double-stranded DNA and low reactivity toward single-stranded DNA. Metal-HAPP complexes with different coordination number and ring size were synthesized and their selectivity and reactivity for DNA bulges were compared. The DNA sequence at the bulge site influences the intensity of cleavage at the bulge and the flanking sites after piperidine treatment. Cleavage specificity of CoII(HAPP)(TFA)2 was characterized extensively using scavenger reagents to quench the cleavage reaction and high-resolution polyacrylamide gel electrophoresis. In addition, 3'-phosphoglycolate cleavage products were trapped and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. These data were used to deduce that the DNA cleavage pathway for CoIIHAPP2+ in the presence of H2O2 involves 4'-H abstraction of the deoxyribose moiety.