RESUMEN
Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Dominios PDZ , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Secuencias de Aminoácidos , Cromatografía , Proteínas de Unión al ADN/química , Humanos , Cinética , Ligandos , Proteínas Oncogénicas Virales/química , Conformación Proteica , Proteoma , Proteínas Represoras/química , Biología de SistemasRESUMEN
BACKGROUND: Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli. RESULTS: The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal. CONCLUSIONS: This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.
Asunto(s)
Endopeptidasas/metabolismo , Escherichia coli/genética , Biosíntesis de Péptidos , Péptidos/genética , Ponzoñas/biosíntesis , Ponzoñas/genética , Animales , Biotecnología/métodos , Clonación Molecular , Disulfuros/química , Endopeptidasas/química , Vectores Genéticos , Ensayos Analíticos de Alto Rendimiento , Oxidación-Reducción , Péptidos/química , Péptidos/aislamiento & purificación , Periplasma/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad , Ponzoñas/química , Ponzoñas/metabolismoRESUMEN
BACKGROUND: Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. RESULTS: In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. CONCLUSIONS: Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs.
Asunto(s)
Escherichia coli/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Biblioteca de Péptidos , Péptidos/genética , Proteínas Recombinantes/aislamiento & purificación , Ponzoñas/genética , Animales , Disulfuros/química , Descubrimiento de Drogas/métodos , Endopeptidasas/metabolismo , Proteínas de Escherichia coli/genética , Oxidación-Reducción , Péptidos/aislamiento & purificación , Péptidos/uso terapéutico , Periplasma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Ponzoñas/químicaRESUMEN
Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
Asunto(s)
Escherichia coli/química , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Ponzoñas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Ponzoñas/biosíntesis , Ponzoñas/genéticaRESUMEN
Hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B constitutes a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. We have screened 17,000 compounds in an enzymatic assay involving the purified NS5B in order to increase the therapeutic arsenal. We hoped to shed some light on the precise mechanism of RNA synthesis. We succeeded in isolating a series of 21 original inhibitors of the RNA synthesis by NS5B. Four of these non-nucleoside inhibitors (NNIs) could be mapped to the known binding site called 'B' as judged by the decrease in their inhibition potency when assayed with a 'B' site mutant, M423T NS5B. Incidentally, our in silico model pointed to Y477 as a key residue for inhibitor binding. In vitro, Y477F mutant loses its sensitivity to the newly discovered inhibitors but is unable to extend primers during the elongation phase. Our results demonstrate that elements of the 'B' site are involved in the conformational changes required in the switch between the different RNA synthesis steps and that compounds targeting this site could lock the enzyme in its initiation phase.