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1.
BMC Immunol ; 24(1): 34, 2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37752417

RESUMO

BACKGROUND: Rapid and accurate diagnosis of individuals with SARS-CoV-2 infection is an effective way to prevent and control the spread of COVID-19. Although the detection of SARS-CoV-2 viral RNA by RT-qPCR is the gold standard for COVID-19 testing, the use of antigen-detecting rapid diagnostic tests (Ag-RDTs) is emerging as a complementary surveillance tool as Omicron case numbers skyrocket worldwide. However, the results from Ag-RDTs are less accurate in individuals with low viral loads. RESULTS: To develop a highly sensitive and accurate Ag-RDT, 90 monoclonal antibodies were raised from guinea pigs immunized with SARS CoV-2 nucleocapsid protein (CoV-2-NP). By applying a capture antibody recognizing the structural epitope of the N-terminal domain of CoV-2-NP and a detection antibody recognizing the C-terminal tail of CoV-2-NP to an automated chemiluminescence flow-through membrane immunoassay device, we developed a novel Ag-RDT, CoV-2-POCube. The CoV-2-POCube exclusively recognizes CoV-2-NP variants but not the nucleocapsid proteins of other human coronaviruses. The CoV-2-POCube achieved a limit of detection sensitivity of 0.20 ~ 0.66 pg/mL of CoV-2-NPs, demonstrating more than 100 times greater sensitivity than commercially available SARS-CoV-2 Ag-RDTs. CONCLUSIONS: CoV-2-POCube has high analytical sensitivity and can detect SARS-CoV-2 variants in 15 min without observing the high-dose hook effect, thus meeting the need for early SARS-CoV-2 diagnosis with lower viral load. CoV-2-POCube is a promising alternative to currently available diagnostic devices for faster clinical decision making in individuals with suspected COVID-19 in resource-limited settings.


Assuntos
Anticorpos Monoclonais , COVID-19 , Humanos , Animais , Cobaias , SARS-CoV-2 , Teste para COVID-19 , COVID-19/diagnóstico , Sensibilidade e Especificidade , Imunoensaio
2.
J Biochem ; 150(1): 73-81, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21478485

RESUMO

Recombinant protein technology is an important tool in many industrial and pharmacological applications. Although the success rate of obtaining soluble proteins is relatively low, knowledge of protein expression/solubility under 'standard' conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we conducted a genome-scale experiment to assess the overexpression and the solubility of human full-length cDNA in an in vivo Escherichia coli expression system and a wheat germ cell-free expression system. We evaluated the influences of sequence and structural features on protein expression/solubility in each system and estimated a minimal set of features associated with them. A comparison of the feature sets related to protein expression/solubility in the in vivo Escherichia coli expression system revealed that the structural information was strongly associated with protein expression, rather than protein solubility. Moreover, a significant difference was found in the number of features associated with protein solubility in the two expression systems.


Assuntos
Sistema Livre de Células/metabolismo , Escherichia coli/metabolismo , Biossíntese de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Triticum/metabolismo , DNA Complementar/genética , Interpretação Estatística de Dados , Escherichia coli/genética , Expressão Gênica , Humanos , Proteínas Recombinantes/isolamento & purificação , Solubilidade , Triticum/genética
3.
Nat Methods ; 5(12): 1011-7, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19054851

RESUMO

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.


Assuntos
Clonagem Molecular/métodos , Genoma Humano/genética , Engenharia de Proteínas/métodos , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/metabolismo , Sistema Livre de Células , Humanos
4.
Extremophiles ; 11(1): 85-94, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16896527

RESUMO

We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdsb encodes a protein of 170 amino acids with an NH(2)-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif, Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH(2)-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins of archaea and bacteria appear to be highly similar in terms of function.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Pyrococcus horikoshii/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Clonagem Molecular , Sequência Conservada , Bases de Dados de Proteínas , Estabilidade Enzimática , Dados de Sequência Molecular , Mutação , Proteína Dissulfeto Redutase (Glutationa)/química , Proteína Dissulfeto Redutase (Glutationa)/genética , Isomerases de Dissulfetos de Proteínas/química , Sinais Direcionadores de Proteínas , Pyrococcus horikoshii/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Temperatura
5.
Proteomics ; 6(1): 54-66, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16287168

RESUMO

Cypoviruses are insect viruses that produce a cytoplasmic crystalline particle called the polyhedron in which progeny virions are occluded. The virion structural protein, VP3, is implicated in the occlusion of viral particles into polyhedra. In this study, we determined the amino acid sequence of VP3 required for occlusion of viral particles into polyhedra and proposed that this sequence could be used as an immobilization signal to direct the stable incorporation of foreign proteins into polyhedra. A large-scale survey revealed that the immobilization signal could, in fact, direct the incorporation of a variety of human proteins into polyhedra. Immune reactivity and protein-protein interactions were detected on the surface of polyhedra containing immobilized foreign proteins, and these particles were shown to be highly stabilized against dehydration. We showed that these particles could be arrayed onto a glass slide by standard spotting and laser manipulation methods. Thus, this approach is well suited for protein expression, purification, and the development of protein microarrays.


Assuntos
Análise Serial de Proteínas , Proteínas/química , Reoviridae/química , Animais , Linhagem Celular , Humanos , Microscopia Confocal , Microscopia Imunoeletrônica , Espectrometria de Fluorescência , Spodoptera
6.
Nucleic Acids Res ; 33(13): e112, 2005 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-16040595

RESUMO

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction-modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5'-GTAC and leave a 3'-TA overhang (5'-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90 degrees C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.


Assuntos
Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Pyrococcus abyssi/enzimologia , Sequência de Bases , Sistema Livre de Células , Biologia Computacional , Enzimas de Restrição do DNA/isolamento & purificação , Genômica , Temperatura Alta , Dados de Sequência Molecular , Extratos Vegetais/química , Biossíntese de Proteínas , Pyrococcus abyssi/genética , Pyrococcus horikoshii/genética , Especificidade por Substrato , Triticum/química
7.
J Mol Biol ; 351(2): 291-8, 2005 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-16019029

RESUMO

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.


Assuntos
Proteínas Arqueais/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Thermococcus/enzimologia , Sequência de Aminoácidos , Animais , Proteínas Arqueais/química , Sítios de Ligação , Bovinos , Cristalografia por Raios X , DNA/química , DNA Polimerase Dirigida por DNA/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase , Conformação Proteica , Estrutura Terciária de Proteína , Eletricidade Estática
8.
Genes Cells ; 9(2): 153-64, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15009092

RESUMO

Specific interactions between transcription factors and cis-acting DNA sequence motifs are primary events for the transcriptional regulation. Many regulatory elements appear to diverge from the most optimal recognition sequences. To evaluate affinities of a transcription factor to various suboptimal sequences, we have developed a new detection method based on the surface plasmon resonance (SPR) imaging technique. Transcription factor MafG and its recognition sequence MARE (Maf recognition elements) were adopted to evaluate the new method. We modified DNA immobilization procedure on to the gold chip, so that a double-stranded DNA array was successfully fabricated. We further found that a hydrophilic flexible spacer composed of the poly (ethylene glycol) moiety between DNA and alkanethiol self-assembled monolayers on the surface is effective for preventing nonspecific adsorption and facilitating specific binding of MafG. Multiple interaction profiles between MafG and six of MARE-related sequences were observed by the SPR imaging technique. The kinetic values obtained by SPR imaging showed very good correlation with those obtained from electrophoretic gel mobility shift assays, although absolute values were deviated from each other. These results demonstrate that the double-stranded DNA array fabricated with the modified multistep procedure can be applied for the comprehensive analysis of the transcription factor-DNA interaction.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Análise Serial de Proteínas/métodos , Proteínas Repressoras/metabolismo , Elementos de Resposta , Ressonância de Plasmônio de Superfície/métodos , Sítios de Ligação , Sequência Consenso , Reagentes de Ligações Cruzadas , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Ouro/química , Humanos , Cinética , Fator de Transcrição MafG , Maleimidas/química , Polietilenoglicóis/química , Proteínas Repressoras/genética , Compostos de Sulfidrila/química
9.
Biol Pharm Bull ; 26(7): 1018-20, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12843630

RESUMO

Penicillin binding proteins (PBPs) are penicillin-sensitive DD-peptidases catalyzing the terminal stages of bacterial cell wall assembly. We identified a Dictyostelium discoideum gene that encodes a protein of 522 amino acids showing similarity to Escherichia coli PBP4. The D. discoideum protein conserves three consensus sequences (SXXK, SXN and KTG) that are responsible for the catalytic activities of PBPs. The gene product prepared in the cell-free translation system showed carboxypeptidase activity but the activity was not detected in the presence of penicillin G. These results demonstrate that the D. discoideum gene encodes a eukaryotic form of penicillin-sensitive carboxypeptidase.


Assuntos
Carboxipeptidases/metabolismo , Dictyosteliida/efeitos dos fármacos , Dictyostelium/efeitos dos fármacos , Penicilinas/farmacologia , Sequência de Aminoácidos/efeitos dos fármacos , Sequência de Aminoácidos/fisiologia , Animais , Sequência de Bases/efeitos dos fármacos , Sequência de Bases/fisiologia , Carboxipeptidases/genética , Dictyosteliida/enzimologia , Dictyostelium/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Dados de Sequência Molecular
10.
Am J Pathol ; 162(2): 381-9, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12547697

RESUMO

In situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3' side. This (AT)10 of the probe is elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes.


Assuntos
HIV-1/isolamento & purificação , Linfonodos/patologia , RNA Mensageiro/isolamento & purificação , Adenina , Adulto , Antígenos Virais/análise , Sequência de Bases , Criança , Pré-Escolar , Sondas de DNA , Feminino , HIV-1/genética , Humanos , Hibridização In Situ/métodos , Lactente , Linfonodos/virologia , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Sensibilidade e Especificidade , Timina
11.
FEBS Lett ; 514(1): 102-5, 2002 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-11904190

RESUMO

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.


Assuntos
Proteínas de Bactérias/genética , Técnicas Genéticas , Bicamadas Lipídicas , Biossíntese de Proteínas , Proteínas de Bactérias/metabolismo , Sistema Livre de Células , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo
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