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1.
Cell ; 152(1-2): 327-39, 2013 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-23332764

RESUMO

Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.


Assuntos
Imunoprecipitação da Cromatina , Modelos Biológicos , Técnica de Seleção de Aptâmeros , Fatores de Transcrição/metabolismo , Animais , DNA/química , Humanos , Cadeias de Markov , Camundongos , Filogenia , Fatores de Transcrição/genética
2.
Proc Natl Acad Sci U S A ; 120(39): e2302500120, 2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37722050

RESUMO

To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen-presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTE) system, a recombinant optogenetics-based molecular tool targeting the T cell receptor (TCR). The LiTE system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation. We established that CD4+ T cells respond to intermittent TCR stimulation more efficiently than their CD8+ T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTE system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTE system provides opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with high spatiotemporal control.


Assuntos
Células Apresentadoras de Antígenos , Linfócitos T CD8-Positivos , Citocinas , Células Epiteliais , Ativação Linfocitária
3.
J Biol Chem ; 298(4): 101784, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35247390

RESUMO

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific analysis for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom-up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins.


Assuntos
Mucinas , Sequência de Aminoácidos , Animais , Glicosilação , Células HEK293 , Humanos , Mucinas/metabolismo , Polissacarídeos/genética , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ovinos
4.
J Cell Sci ; 134(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34350965

RESUMO

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.


Assuntos
Citoesqueleto , Septinas , Actinas , Animais , Citoesqueleto/metabolismo , Humanos , Mamíferos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Septinas/genética , Septinas/metabolismo
5.
Chembiochem ; 23(24): e202200595, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36269004

RESUMO

In 2019 four groups reported independently the development of a simplified enzymatic access to the diphosphates (IPP and DMAPP) of isopentenol and dimethylallyl alcohol (IOH and DMAOH). The former are the two universal precursors of all terpenes. We report here on an improved version of what we call the terpene mini-path as well as its use in enzymatic cascades in combination with various transferases. The goal of this study is to demonstrate the in vitro utility of the TMP in, i) synthesizing various natural terpenes, ii) revealing the product selectivity of an unknown terpene synthase, or iii) generating unnatural cyclobutylated terpenes.


Assuntos
Alquil e Aril Transferases , Terpenos , Transferases , Difosfatos
6.
PLoS Biol ; 17(6): e3000317, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181075

RESUMO

Within the cytoplasm of a single cell, several actin networks can coexist with distinct sizes, geometries, and protein compositions. These actin networks assemble in competition for a limited pool of proteins present in a common cellular environment. To predict how two distinct networks of actin filaments control this balance, the simultaneous assembly of actin-related protein 2/3 (Arp2/3)-branched networks and formin-linear networks of actin filaments around polystyrene microbeads was investigated with a range of actin accessory proteins (profilin, capping protein, actin-depolymerizing factor [ADF]/cofilin, and tropomyosin). Accessory proteins generally affected actin assembly rates for the distinct networks differently. These effects at the scale of individual actin networks were surprisingly not always correlated with corresponding loss-of-function phenotypes in cells. However, our observations agreed with a global interpretation, which compared relative actin assembly rates of individual actin networks. This work supports a general model in which the size of distinct actin networks is determined by their relative capacity to assemble in a common and competing environment.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/fisiologia , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Animais , Humanos , Cinética , Proteínas dos Microfilamentos/metabolismo , Profilinas/metabolismo , Mapas de Interação de Proteínas/fisiologia , Tropomiosina
7.
Proc Natl Acad Sci U S A ; 116(13): 6063-6068, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850540

RESUMO

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two of which were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.


Assuntos
Metabolismo dos Carboidratos , Enzimas/genética , Análise de Sequência de Proteína , Sequência de Aminoácidos , Animais , Metabolismo dos Carboidratos/genética , Enzimas/metabolismo , Genômica/métodos , Humanos , Polissacarídeos/metabolismo , Análise de Sequência de DNA , Relação Estrutura-Atividade
8.
Int J Mol Sci ; 23(7)2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35409382

RESUMO

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.


Assuntos
Escherichia coli , Plantas , Biomassa , Carboidratos , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Plantas/genética , Plantas/metabolismo
9.
J Biol Chem ; 294(37): 13755-13768, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31346033

RESUMO

Protection of neuronal homeostasis is a major goal in the management of neurodegenerative diseases. Microtubule-associated Ser/Thr kinase 2 (MAST2) inhibits neurite outgrowth, and its inhibition therefore represents a potential therapeutic strategy. We previously reported that a viral protein (G-protein from rabies virus) capable of interfering with protein-protein interactions between the PDZ domain of MAST2 and the C-terminal moieties of its cellular partners counteracts MAST2-mediated suppression of neurite outgrowth. Here, we designed peptides derived from the native viral protein to increase the affinity of these peptides for the MAST2-PDZ domain. Our strategy involved modifying the length and flexibility of the noninteracting sequence linking the two subsites anchoring the peptide to the PDZ domain. Three peptides, Neurovita1 (NV1), NV2, and NV3, were selected, and we found that they all had increased affinities for the MAST2-PDZ domain, with Kd values decreasing from 1300 to 60 nm, while target selectivity was maintained. A parallel biological assay evaluating neurite extension and branching in cell cultures revealed that the NV peptides gradually improved neural activity, with the efficacies of these peptides for stimulating neurite outgrowth mirroring their affinities for MAST2-PDZ. We also show that NVs can be delivered into the cytoplasm of neurons as a gene or peptide. In summary, our findings indicate that virus-derived peptides targeted to MAST2-PDZ stimulate neurite outgrowth in several neuron types, opening up promising avenues for potentially using NVs in the management of neurodegenerative diseases.


Assuntos
Neuritos/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Domínios PDZ/fisiologia , Estimulantes do Sistema Nervoso Central/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Microtúbulos/metabolismo , Neurônios/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Vírus da Raiva , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia
10.
Nucleic Acids Res ; 46(D1): D718-D725, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29149270

RESUMO

ANISEED (www.aniseed.cnrs.fr) is the main model organism database for tunicates, the sister-group of vertebrates. This release gives access to annotated genomes, gene expression patterns, and anatomical descriptions for nine ascidian species. It provides increased integration with external molecular and taxonomy databases, better support for epigenomics datasets, in particular RNA-seq, ChIP-seq and SELEX-seq, and features novel interactive interfaces for existing and novel datatypes. In particular, the cross-species navigation and comparison is enhanced through a novel taxonomy section describing each represented species and through the implementation of interactive phylogenetic gene trees for 60% of tunicate genes. The gene expression section displays the results of RNA-seq experiments for the three major model species of solitary ascidians. Gene expression is controlled by the binding of transcription factors to cis-regulatory sequences. A high-resolution description of the DNA-binding specificity for 131 Ciona robusta (formerly C. intestinalis type A) transcription factors by SELEX-seq is provided and used to map candidate binding sites across the Ciona robusta and Phallusia mammillata genomes. Finally, use of a WashU Epigenome browser enhances genome navigation, while a Genomicus server was set up to explore microsynteny relationships within tunicates and with vertebrates, Amphioxus, echinoderms and hemichordates.


Assuntos
Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Genoma , Urocordados/genética , Animais , Evolução Biológica , Ciona intestinalis/genética , DNA/metabolismo , Mineração de Dados , Evolução Molecular , Expressão Gênica , Ontologia Genética , Internet , Anotação de Sequência Molecular , Filogenia , Ligação Proteica , Especificidade da Espécie , Fatores de Transcrição/metabolismo , Transcrição Gênica , Vertebrados/genética , Navegador
11.
Mol Microbiol ; 110(5): 777-795, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30204278

RESUMO

With increasing numbers of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and associated functionality of phage adhesion devices. Recently, decorations have been reported in the tail and neck passage structures of members of the so-called 936 group of lactococcal siphophages. In the current report, using bioinformatic analysis we identified a conserved carbohydrate binding module (CBM) among many of the virion baseplate Dit components, in addition to the CBM present in the 'classical' receptor binding proteins (RBPs). We observed that, within these so-called 'evolved' Dit proteins, the identified CBMs have structurally conserved folds, yet can be grouped into four distinct classes. We expressed such modules in fusion with GFP, and demonstrated their binding capability to their specific host using fluorescent binding assays with confocal microscopy. We detected evolved Dits in several phages infecting various Gram-positive bacterial species, including mycobacteria. The omnipresence of CBM domains in siphophages indicates their auxiliary role in infection, as they can assist in the specific recognition of and attachment to their host, thus ensuring a highly efficient and specific phage-host adhesion process as a prelude to DNA injection.


Assuntos
Lactococcus lactis/virologia , Siphoviridae/genética , Siphoviridae/metabolismo , Proteínas da Cauda Viral/genética , Vírion/genética , Carboidratos/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica
12.
Environ Microbiol ; 20(1): 228-240, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29076618

RESUMO

The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.


Assuntos
Flagelos/metabolismo , Flagelina/metabolismo , Glicosiltransferases/genética , Magnetospirillum/metabolismo , Proteínas de Bactérias , Campylobacter jejuni/enzimologia , Flagelos/genética , Glicosilação , Magnetospirillum/enzimologia , Magnetospirillum/genética , Ácidos Siálicos/química , Açúcares Ácidos/metabolismo
13.
Nat Methods ; 12(8): 787-93, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26053890

RESUMO

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.


Assuntos
Ensaios de Triagem em Larga Escala , Domínios PDZ , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Motivos de Aminoácidos , Cromatografia , Proteínas de Ligação a DNA/química , Humanos , Cinética , Ligantes , Proteínas Oncogênicas Virais/química , Conformação Proteica , Proteoma , Proteínas Repressoras/química , Biologia de Sistemas
14.
Microb Cell Fact ; 16(1): 6, 2017 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-28095880

RESUMO

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.


Assuntos
Escherichia coli/genética , Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Peptídeos/genética , Proteínas Recombinantes/isolamento & purificação , Peçonhas/genética , Animais , Dissulfetos/química , Descoberta de Drogas/métodos , Endopeptidases/metabolismo , Proteínas de Escherichia coli/genética , Oxirredução , Peptídeos/isolamento & purificação , Peptídeos/uso terapêutico , Periplasma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Peçonhas/química
15.
Microb Cell Fact ; 16(1): 4, 2017 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-28093085

RESUMO

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.


Assuntos
Endopeptidases/metabolismo , Escherichia coli/genética , Biossíntese Peptídica , Peptídeos/genética , Peçonhas/biossíntese , Peçonhas/genética , Animais , Biotecnologia/métodos , Clonagem Molecular , Dissulfetos/química , Endopeptidases/química , Vetores Genéticos , Ensaios de Triagem em Larga Escala , Oxirredução , Peptídeos/química , Peptídeos/isolamento & purificação , Periplasma/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Peçonhas/química , Peçonhas/metabolismo
16.
BMC Biotechnol ; 16(1): 86, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27905914

RESUMO

BACKGROUND: Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. RESULTS: Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. CONCLUSIONS: An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.


Assuntos
Genes Sintéticos/genética , Ensaios de Triagem em Larga Escala/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Venenos de Serpentes/genética , Algoritmos , Animais , Técnicas de Cultura Celular por Lotes/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Peso Molecular , Serpentes
17.
Adv Exp Med Biol ; 896: 43-58, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27165318

RESUMO

Single protein expression technologies have strongly benefited from the Structural Genomics initiatives that have introduced parallelization at the laboratory level. Specifically, the developments made in the wake of these initiatives have revitalized the use of Escherichia coli as major host for heterologous protein expression. In parallel to these improvements for single expression, technologies for complex reconstitution by co-expression in E. coli have been developed. Assessments of these co-expression technologies have highlighted the need for combinatorial experiments requiring automated protocols. These requirements can be fulfilled by adapting the high-throughput approaches that have been developed for single expression to the co-expression technologies. Yet, challenges are laying ahead that further need to be addressed and that are only starting to be taken into account in the case of single expression. These notably include the biophysical characterization of the samples at the small-scale level. Specifically, these approaches aim at discriminating the samples at an early stage of their production based on various biophysical criteria leading to cost-effectiveness and time-saving. This chapter addresses these various issues to provide the reader with a broad and comprehensive overview of complex reconstitution and characterization by co-expression in E. coli.


Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/metabolismo , Técnicas de Transferência de Genes , Ensaios de Triagem em Larga Escala , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Animais , Automação Laboratorial , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Humanos , Complexos Multiproteicos , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-Atividade
18.
Nucleic Acids Res ; 40(7): 3245-58, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22140099

RESUMO

Besides their commonly attributed role in the maintenance of low-copy number plasmids, toxin/antitoxin (TA) loci, also called 'addiction modules', have been found in chromosomes and associated to a number of biological functions such as: reduction of protein synthesis, gene regulation and retardation of cell growth under nutritional stress. The recent discovery of TA loci in obligatory intracellular species of the Rickettsia genus has prompted new research to establish whether they work as stress response elements or as addiction systems that might be toxic for the host cell. VapBC2 is a TA locus from R. felis, a pathogen responsible for flea-borne spotted fever in humans. The VapC2 toxin is a PIN-domain protein, whereas the antitoxin, VapB2, belongs to the family of swapped-hairpin ß-barrel DNA-binding proteins. We have used a combination of biophysical and structural methods to characterize this new toxin/antitoxin pair. Our results show how VapB2 can block the VapC2 toxin. They provide a first structural description of the interaction between a swapped-hairpin ß-barrel protein and DNA. Finally, these results suggest how the VapC2/VapB2 molar ratio can control the self-regulation of the TA locus transcription.


Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , DNA Bacteriano/química , Rickettsia felis/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , DNA Bacteriano/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas
19.
IUCrJ ; 11(Pt 3): 374-383, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38656310

RESUMO

The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors.


Assuntos
Antivirais , Endonucleases , Vírus La Crosse , Triazinas , Vírus La Crosse/efeitos dos fármacos , Vírus La Crosse/enzimologia , Antivirais/farmacologia , Antivirais/química , Endonucleases/antagonistas & inibidores , Endonucleases/metabolismo , Endonucleases/química , Dibenzotiepinas , Morfolinas/farmacologia , Morfolinas/química , Piridonas/farmacologia , Piridonas/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Animais , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/metabolismo
20.
FEBS J ; 291(7): 1439-1456, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38129294

RESUMO

We report here the identification, characterization and three-dimensional (3D) structure determination of NaNga, a newly identified ß-N-acetylgalactosaminidase from the Gram-negative soil bacterium Niabella aurantiaca DSM 17617. When recombinantly expressed in Escherichia coli, the enzyme selectively cleaved 4-nitrophenyl-N-acetyl-ß-d-galactosamine (pNP-ß-d-GalpNAc). The X-ray crystal structure of the protein was refined to 2.5 Å and consists of an N-terminal ß-sandwich domain and a (ß/α)8 barrel catalytic domain. Despite a mere 22% sequence identity, the 3D structure of NaNga is similar to those previously determined for family GH123 members, suggesting it also employs the same substrate-assisted catalytic mechanism. Inhibition by N-acetyl-galactosamine thiazoline (GalNAc-thiazoline) supports the suggested mechanism. A phylogenetic analysis of its proximal sequence space shows significant clustering of unknown sequences around NaNga with sufficient divergence with previously identified GH123 members to subdivide this family into distinct subfamilies. Although the actual biological substrate of our enzyme remains unknown, examination of the active site pocket suggests that it may be a ß-N-acetylgalactosaminide substituted by a monosaccharide at O-3. Analysis of the genomic context suggests, in turn, that this substituted ß-N-acetylgalactosaminide may be appended to a d-arabinan from an environmental Actinomycete.


Assuntos
Bacteroidetes , Galactosamina , beta-N-Acetil-Galactosaminidase , Filogenia , Domínio Catalítico , Especificidade por Substrato
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