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1.
Med Sci (Paris) ; 36(8-9): 797-802, 2020.
Artigo em Francês | MEDLINE | ID: mdl-32755538

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2, which emerged in China at the end of 2019, is responsible for a global health crisis resulting in the confinement of more than 3 billion people worldwide and the sharp decline of the world economy. In this context, a race against the clock is launched in order to develop a treatment to stop the pandemic as soon as possible. A study published in Nature by the Volker Thiel team reports the development of reverse genetics for SARS-CoV-2 allowing them to recreate the virus in just a few weeks. The perspectives of this work are very interesting since it will allow the genetic manipulation of the virus and thus the development of precious tools which will be useful to fight the infection. Even though this approach represents a technological leap that will improve our knowledge of the virus, it also carries the germ of possible misuse and the creation of the virus for malicious purposes. The advantages and disadvantages of recreating SARS-CoV-2 in this pandemic period are discussed in this mini-synthesis.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Organismos Geneticamente Modificados , Pandemias , Pneumonia Viral/virologia , Genética Reversa/métodos , Betacoronavirus/patogenicidade , Derramamento de Material Biológico , Cromossomos Artificiais de Levedura , Clonagem Molecular/métodos , Coronaviridae/classificação , Coronaviridae/genética , Coronaviridae/patogenicidade , Infecções por Coronavirus/prevenção & controle , DNA Complementar/genética , Especificidade de Hospedeiro , Humanos , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/patogenicidade , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , RNA Viral/genética , Recombinação Genética , Risco , Vacinas Virais
2.
Gene ; 761: 145043, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32777530

RESUMO

Tonoplast Intrinsic Proteins (TIPs) constitute a significant class of the aquaporins. The TIPs control water trade among cytosolic and vacuolar compartments and can also transport glycerol, ammonia, urea, hydrogen peroxide, metals/metalloids, and so forth. Additionally, TIPs are engaged with different abiotic stress responses and developmental processes like leaf expansion, root elongation and seed germination. In this study, ten TIP genes in the rice genome were identified from Oryza sativa ssp indica. Among these, representative groups of TIP genes were cloned and sequenced whilst some TIP sequences showed stop codons in the coding region. The secondary structure analysis represented six conserved transmembrane helices along with the inter-helical regions having conserved motifs. The representative three-dimensional tetrameric design of protein sequence of TIP1;1 displayed key features like NPA motifs, aromatic/arginine (ar/R) selectivity filters, and Froger's residues. The vacuolar localization, transmembrane topological properties, and conserved motif analysis of the cloned genes altogether supported their identity as TIPs. An unrooted phylogenetic tree delineated the relatedness of TIPs from Oryza with different species and bunched them into five clades. The promoter analysis uncovered key regulons associated with administering abiotic stress responses. Gene expression studies showed thatTIPsare differentially regulated under salt and drought stress at various time points in shoots and roots of rice. Also, the pattern of expression was found to be significantly variable in five different rice tissues. The heat-map based tissue and stress- specific expression analysis supported the experimental findings. In conclusion, the identification and transcript-level expression studies of TIPs significantly contribute towards the comprehension of their utilitarian significance in the abiotic stress response.


Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Aquaporinas/genética , Aquaporinas/metabolismo , Clonagem Molecular/métodos , Secas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Oryza/metabolismo , Filogenia , Folhas de Planta/metabolismo , Cloreto de Sódio/metabolismo , Estresse Fisiológico/genética , Vacúolos/genética , Água/metabolismo
3.
PLoS One ; 15(7): e0235853, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701967

RESUMO

PCR-based amplification of annotated genes has allowed construction of expression clones at genome-scale using classical and recombination-based cloning technologies. However, genome-scale expression and purification of proteins for down-stream applications is often limited by challenges such as poor expression, low solubility, large size of multi-domain proteins, etc. Alternatively, DNA fragment libraries in expression vectors can serve as the source of protein fragments with each fragment encompassing a function of its whole protein counterpart. However, the random DNA fragmentation and cloning result in only 1 out of 18 clones being in the correct open-reading frame (ORF), thus, reducing the overall efficiency of the system. This necessitates the selection of correct ORF before expressing the protein fragments. This paper describes a highly efficient ORF selection system for DNA fragment libraries, which is based on split beta-lactamase protein fragment complementation. The system has been designed to allow seamless transfer of selected DNA fragment libraries into any downstream vector systems using a restriction enzyme-free cloning strategy. The strategy has been applied for the selection of ORF using model constructs to show near 100% selection of the clone encoding correct ORF. The system has been further validated by construction of an ORF-selected DNA fragment library of 30 genes of M. tuberculosis. Further, we have successfully demonstrated the cytosolic expression of ORF-selected protein fragments in E. coli.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Teste de Complementação Genética/métodos , Fases de Leitura Aberta , beta-Lactamases/genética , Proteínas de Bactérias/metabolismo , Escherichia coli , Biblioteca Gênica , Mycobacterium tuberculosis , beta-Lactamases/metabolismo
4.
Nucleic Acids Res ; 48(13): 7483-7501, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32510132

RESUMO

The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, we developed vitRIP, an unbiased, transcriptome-wide in vitro assay that reveals RNA binding specificity. We found that MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.


Assuntos
Montagem e Desmontagem da Cromatina , RNA Longo não Codificante/metabolismo , Transcriptoma , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular/métodos , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ligação Proteica , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Nucleic Acids Res ; 48(13): e76, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32479612

RESUMO

The control of gene expression noise is important for improving drug treatment and the performance of synthetic biological systems. Previous work has tuned gene expression noise by changing the rate of transcription initiation, mRNA degradation, and mRNA translation. However, these methods are invasive: they require changes to the target genetic components. Here, we create an orthogonal system based on CRISPR-dCas9 to tune gene expression noise. Specifically, we modulate the gene expression noise of a reporter gene in Escherichia coli by incorporating CRISPR activation and repression (CRISPRar) simultaneously in a single cell. The CRISPRar uses a single dCas9 that recognizes two different single guide RNAs (sgRNA). We build a library of sgRNA variants with different expression activation and repression strengths. We find that expression noise and mean of a reporter gene can be tuned independently by CRISPRar. Our results suggest that the expression noise is tuned by the competition between two sgRNAs that modulate the binding of RNA polymerase to promoters. The CRISPRar may change how we tune expression noise at the genomic level. Our work has broad impacts on the study of gene functions, phenotypical heterogeneity, and genetic circuit control.


Assuntos
Sistemas CRISPR-Cas/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Expressão Gênica/genética , RNA Guia/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter/genética
6.
PLoS One ; 15(5): e0232959, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401802

RESUMO

The elucidation of mechanisms behind the thermostability of proteins is extremely important both from the theoretical and applied perspective. Here we report the crystal structure of methylenetetrahydrofolate dehydrogenase (MTHFD) from Thermus thermophilus HB8, a thermophilic model organism. Molecular dynamics trajectory analysis of this protein at different temperatures (303 K, 333 K and 363 K) was compared with homologous proteins from the less temperature resistant organism Thermoplasma acidophilum and the mesophilic organism Acinetobacter baumannii using several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis. These methods enabled the determination of important residues for the thermostability of this enzyme. The description of rotamer distributions by Gini coefficients and Kullback-Leibler (KL) divergence both revealed significant correlations with temperature. The emerging view seems to indicate that a static salt bridge/charged residue network plays a fundamental role in the temperature resistance of Thermus thermophilus MTHFD by enhancing both electrostatic interactions and entropic energy dispersion. Furthermore, this analysis uncovered a relationship between residue mutations and evolutionary pressure acting on thermophilic organisms and thus could be of use for the design of future thermostable enzymes.


Assuntos
Clonagem Molecular/métodos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/química , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Thermus thermophilus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Estabilidade Enzimática , Modelos Moleculares , Simulação de Dinâmica Molecular , Análise de Componente Principal , Estrutura Secundária de Proteína , Termodinâmica , Thermus thermophilus/genética
7.
Nature ; 582(7813): 561-565, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32365353

RESUMO

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.


Assuntos
Betacoronavirus/genética , Clonagem Molecular/métodos , Infecções por Coronavirus/virologia , Genoma Viral/genética , Genômica/métodos , Pneumonia Viral/virologia , Genética Reversa/métodos , Biologia Sintética/métodos , Animais , China/epidemiologia , Chlorocebus aethiops , Cromossomos Artificiais de Levedura/metabolismo , Infecções por Coronavirus/epidemiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular , Humanos , Mutação , Pandemias/estatística & dados numéricos , Pneumonia Viral/epidemiologia , Vírus Sinciciais Respiratórios/genética , Saccharomyces cerevisiae/genética , Células Vero , Proteínas Virais/metabolismo , Zika virus/genética
8.
Nucleic Acids Res ; 48(9): 5169-5182, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32246719

RESUMO

One primary objective of synthetic biology is to improve the sustainability of chemical manufacturing. Naturally occurring biological systems can utilize a variety of carbon sources, including waste streams that pose challenges to traditional chemical processing, such as lignin biomass, providing opportunity for remediation and valorization of these materials. Success, however, depends on identifying micro-organisms that are both metabolically versatile and engineerable. Identifying organisms with this combination of traits has been a historic hindrance. Here, we leverage the facile genetics of the metabolically versatile bacterium Acinetobacter baylyi ADP1 to create easy and rapid molecular cloning workflows, including a Cas9-based single-step marker-less and scar-less genomic integration method. In addition, we create a promoter library, ribosomal binding site (RBS) variants and test an unprecedented number of rationally integrated bacterial chromosomal protein expression sites and variants. At last, we demonstrate the utility of these tools by examining ADP1's catabolic repression regulation, creating a strain with improved potential for lignin bioprocessing. Taken together, this work highlights ADP1 as an ideal host for a variety of sustainability and synthetic biology applications.


Assuntos
Acinetobacter/genética , Engenharia Metabólica , Acinetobacter/metabolismo , Clonagem Molecular/métodos , Genoma Bacteriano , Genômica , Lignina/metabolismo , Regiões Promotoras Genéticas , Ribossomos/metabolismo
9.
Nucleic Acids Res ; 48(8): 4139-4146, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32232356

RESUMO

GoldenBraid is a rapid, modular, and robust cloning system used to assemble and combine genetic elements. Dictyostelium amoebae represent an intriguing synthetic biological chassis with tractable applications in development, chemotaxis, bacteria-host interactions, and allorecognition. We present GoldenBraid as a synthetic biological framework for Dictyostelium, including a library of 250 DNA parts and assemblies and a proof-of-concept strain that illustrates cAMP-chemotaxis with four fluorescent reporters coded by one plasmid.


Assuntos
Clonagem Molecular/métodos , Dictyostelium/genética , Quimiotaxia , AMP Cíclico/fisiologia , Dictyostelium/fisiologia , Proteínas Luminescentes/genética , Biologia Sintética/métodos
10.
Hum Genet ; 139(10): 1233-1246, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32277284

RESUMO

Approximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable. Unfortunately, cloning repeat expansions is often challenging and presents a significant bottleneck to practical investigation. Here, we describe a clear method for seamless and systematic cloning of practically any microsatellite repeat expansion. We use cloning and expansion of GGGGCC repeats, which are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as an example. We employ a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors. We describe methods to validate repeat expansion cloning, including diagnostic restriction digestion, PCR across the repeat, and next-generation long-read MinION nanopore sequencing. Validated cloning of microsatellite repeats beyond the critical expansion threshold can facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Clonagem Molecular/métodos , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Repetições de Microssatélites , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Sequência de Bases , Proteína C9orf72/metabolismo , Enzimas de Restrição do DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Humano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
Microb Cell Fact ; 19(1): 63, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32156270

RESUMO

BACKGROUND: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas9 system. RESULTS: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. CONCLUSIONS: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Edição de Genes/métodos , Plasmídeos/genética , RNA Guia/genética , Recombinação Genética , Deleção de Sequência
12.
PLoS One ; 15(3): e0229216, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134955

RESUMO

RNA biogenesis and mRNA transport are an intricate process for every eukaryotic cell. SAGA, a transcriptional coactivator and TREX-2 are the two major complexes participate in this process. Sus1 is a transcription export factor and part of both the SAGA and the TREX-2 complex. The competitive exchange of Sus1 molecule between SAGA and TREX-2 complex modulates their function which is credited to structural plasticity of Sus1. Here, we portray the biophysical characterization of Sus1 from S. cerevisiae. The recombinant Sus1 is a α-helical structure which is stable at various pH conditions. We reported the α-helix to ß-sheet transition at the low pH as well as at high pH. Sus1 showed 50% reduction in the fluorescence intensity at pH-2 as compared to native protein. The fluorescence studies demonstrated the unfolding of tertiary structure of the protein with variation in pH as compared to neutral pH. The same results were obtained in the ANS binding and acrylamide quenching studies. Similarly, the secondary structure of the Sus1 was found to be stable till 55% alcohol concentration while tertiary structure was stable up to 20% alcohol concentration. Further increase in the alcohol concentration destabilizes the secondary as well as tertiary structure. The 300 mM concentration of ammonium sulfate also stabilizes the secondary structure of the protein. The structural characterization of this protein is expected to unfold the process of the transportation of the mRNA with cooperation of different proteins.


Assuntos
Clonagem Molecular/métodos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sulfato de Amônio/farmacologia , Concentração de Íons de Hidrogênio , Modelos Moleculares , Proteínas Nucleares/metabolismo , Ligação Proteica , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Desdobramento de Proteína , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Arch Microbiol ; 202(6): 1449-1458, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32189018

RESUMO

Polymerases are enzymes that synthesize long chains or polymers of nucleic acids including DNA or RNA from nucleotides. They assemble nucleic acids by copying a DNA or RNA template strand using base-pairing interactions. One of the polymerase enzymes, Taq DNA polymerase, originally isolated from Thermus aquaticus (Taq) is a widely used enzyme in molecular biology so far. The thermostable properties of this enzyme have contributed majorly to the specificity, automation, and efficacy of the polymerase chain reaction (PCR), making it a powerful tool for today's molecular biology researches across the globe. The purification of Taq DNA polymerase from the native host results in low yield, more labor and time consumption. Therefore, many studies have been previously conducted to obtain this enzyme using alternative hosts. So far, all the existing methodologies are more laborious, time-consuming and require heavy expense. We used a novel approach to purify the enzyme with relatively high efficiency, yield and minimum time consumption using Escherichia coli (E. coli) as an alternative host. We cloned a 2500 base pair Taq DNA polymerase gene into pGEX-4T-1 vector, containing a GST-tag, downstream of tac promoter and overexpressed it using isopropyl ß-d-1-thiogalactopyranoside (IPTG) as an inducer. The enzyme was efficiently purified using novel chromatography approaches and was used in routine PCR assays in our laboratory. Our findings suggest a novel approach to facilitate the availability of polymerases for molecular and diagnostic studies. In the future, it may be used for the purification of other recombinant peptides or proteins used in structural biology and proteomics-based researches.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/enzimologia , Taq Polimerase/genética , Taq Polimerase/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica/genética , Nucleotídeos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Taq Polimerase/química
14.
Protein Expr Purif ; 169: 105586, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32001358

RESUMO

Eukaryotic recombinant proteins expressed in bacterial cells usually aggregate within the cells as inclusion bodies. Despite the widely-accepted theory considering inclusion bodies as inactive materials, inclusion bodies may contain large amounts of correctly-folded active recombinant proteins. Proteins trapped in inclusion bodies can be released using a high pH solution (pH ≥ 11); however, they may undergo structural changes in such pH conditions that may lead to their inactivation. Shifting in pH alongside the use of metal ions can help recover protein activity. The model protein we used in this study, 9R-Nimo.scFv, is highly active when extracted from bacterial inclusion bodies at high pH condition (pH 12) but loses its activity when pH is reduced to pH 7. We evaluated the capacity of nine salt solutions in terms of recovering protein activity in neutral pH conditions and found that ZnSO4 solution was the best one for this purpose. KNO3 and MnSO4 were also found to have a good capacity for recovering protein activity, as well.


Assuntos
Corpos de Inclusão/química , Íons/análise , Proteínas Recombinantes/química , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Íons/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteínas Recombinantes/biossíntese
15.
Protein Expr Purif ; 169: 105587, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32001359

RESUMO

Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks - purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation. Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state. The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.


Assuntos
Fosforribosil Pirofosfato/biossíntese , Proteínas Recombinantes de Fusão , Cromatografia de Afinidade , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Fosforribosil Pirofosfato/química , Fosforribosil Pirofosfato/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Temperatura
16.
Curr Opin Cell Biol ; 63: 114-124, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32058267

RESUMO

Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.


Assuntos
Clonagem Molecular , Proteínas Luminescentes/genética , Optogenética/métodos , Optogenética/tendências , Transdução de Sinais/genética , Animais , Técnicas Biossensoriais , Rastreamento de Células/métodos , Rastreamento de Células/tendências , Clonagem Molecular/métodos , Humanos , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Imagem Molecular/tendências , Técnicas de Rastreamento Neuroanatômico/métodos , Técnicas de Rastreamento Neuroanatômico/tendências
17.
PLoS One ; 15(2): e0228112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040512

RESUMO

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.


Assuntos
Engenharia Celular/métodos , Clonagem Molecular/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Expressão Gênica , Biblioteca Gênica , Genes MHC Classe I/genética , Genes MHC da Classe II/genética , Células HEK293 , Humanos , Cinética , Receptores de Antígenos de Linfócitos T alfa-beta/genética
18.
Gene ; 736: 144421, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32018014

RESUMO

5-Aminolevulinic acid synthase (ALAS) is the rate-limiting enzyme in the biosynthesis of heme, a prosthetic group that is found in hemoproteins, including those involved in molting. To better understand the roles of ALAS in L. vannamei (LvALAS), we analyzed its sequence and tissue distribution, the effects of age and bacterial infection on its gene expression, and the effects of LvALAS gene silencing. We also examined the expressions of three hemoproteins, the cytochrome oxidase subunit I (COX I) and subunit IV (COX IV) and catalase. Three LvALAS splicing variants were found in the hepatopancreas, with the main splicing variant having an open reading frame that encodes 532 aa. LvALAS transcripts were found in each of the eleven tissues tested in this study, with the highest gene expression in the intestine. The transcript abundances of LvALAS, COX I and COX IV in the hepatopancreas and stomach tended to decrease with age. LvALAS and catalase gene expressions significantly increased in the stomach after V. parahaemolyticus infection. LvALAS gene expression in the hepatopancreas, stomach and intestine (12- and 24-hours post-injection) was relatively lower in dsALAS-injected shrimp than in PBS-injected shrimp. All the PBS-injected shrimp molted after 8-10 days while no molting activity was observed in the dsALAS-injected shrimp group within the 14 days post-injection period. Our results provide evidence that (1) only the housekeeping form of ALAS exists in L. vannamei; LvALAS gene expression (2) decreases with age and (3) increases after bacterial infection; and (4) an ALAS-dependent pathway is necessary for proper molting in L. vannamei.


Assuntos
5-Aminolevulinato Sintetase/genética , Proteínas de Artrópodes/genética , Expressão Gênica/genética , Penaeidae/genética , Sequência de Aminoácidos , Ácido Aminolevulínico/metabolismo , Animais , Clonagem Molecular/métodos , Hepatopâncreas/metabolismo , Hepatopâncreas/patologia , Intestinos/patologia , Penaeidae/patogenicidade , Filogenia , Alinhamento de Sequência , Estômago/patologia
19.
Nat Protoc ; 15(3): 734-749, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32005981

RESUMO

Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.


Assuntos
Clonagem Molecular/métodos , DNA Fúngico/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas , Replicação do DNA , Vetores Genéticos , Plasmídeos , Origem de Replicação , Transformação Genética
20.
Protein Expr Purif ; 169: 105568, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31935447

RESUMO

About half a century after antibiotics discovery, multi-antibiotic-resistant bacteria posed a new challenge to medicine. Attempts to discover new antibiotics have drawn the attention to Antimicrobial Peptides (AMPs). The rapid growth, besides its known genetic and manipulation systems, makes E. coli the preferred host system for production of recombinant proteins on an industrial scale. To produce AMPs in E. coli, the application of fusion-tags with the aim of stability, solubility, and prevention of antimicrobial activity is one of the best practices in this regard. In this study, we presented two different expression systems for the production of PR-39 in E. coli; one in fusion with intein-Chitin binding domain (CBD) and another in fusion with SUMO accompanied by polyhistidine affinity tag. Both were cloned in the NdeI-XhoI sites of pET-17b and transformed to E. coli BL21 (DE3) pLysS. Recombinant bacteria were cultured and induced with 0.4 mM IPTG at 30 °C. Expression and purification of target proteins were confirmed by Tricine- SDS-PAGE and dot blot analysis. Recovery of 250 µg PR-39/L from SUMO fusion system and 280 µg PR-39/L from the intein fusion system was achieved. Both purified peptides showed antibacterial activity using MIC/MBC demonstrating their functionality after SUMO and intein mediated purification.


Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Escherichia coli , Proteínas Recombinantes de Fusão/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Inteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética
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