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1.
Protein & Cell ; (12): 877-888, 2021.
Article in English | WPRIM | ID: wpr-922482

ABSTRACT

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M


Subject(s)
Antiviral Agents/therapeutic use , Binding Sites , COVID-19/virology , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Drug Evaluation, Preclinical , Drug Repositioning , High-Throughput Screening Assays/methods , Humans , Imidazoles/therapeutic use , Inhibitory Concentration 50 , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Naphthoquinones/therapeutic use , Protease Inhibitors/therapeutic use , Protein Structure, Tertiary , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification
2.
Braz. j. microbiol ; 49(2): 414-421, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-889234

ABSTRACT

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.


Subject(s)
Antifungal Agents/pharmacology , Chitinases/pharmacology , Hordeum/enzymology , Recombinant Proteins/metabolism , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Blotting, Western , Chitinases/chemistry , Chitinases/genetics , Chitinases/isolation & purification , Chromatography, Affinity , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hordeum/genetics , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid
3.
Braz. j. microbiol ; 48(4): 809-814, Oct.-Dec. 2017. graf
Article in English | LILACS | ID: biblio-889176

ABSTRACT

ABSTRACT This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5 µM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 µM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics.


Subject(s)
Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Bacillus subtilis/genetics , Genetic Vectors/genetics , Insect Proteins/genetics , Insect Proteins/metabolism , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/pharmacology , Bacillus subtilis/metabolism , Escherichia coli/drug effects , Escherichia coli/growth & development , Gene Expression , Genetic Vectors/metabolism , Insect Proteins/isolation & purification , Insect Proteins/pharmacology , Protein Engineering , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology
4.
Electron. j. biotechnol ; 19(4): 81-89, July 2016. ilus
Article in English | LILACS | ID: lil-793957

ABSTRACT

Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and >98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.


Subject(s)
Saccharomyces cerevisiae/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/biosynthesis , Antithrombin III/isolation & purification , Antithrombin III/biosynthesis , Blotting, Western , Chromatography, High Pressure Liquid , Bioreactors , Fermentation , Filtration
5.
Braz. j. microbiol ; 46(1): 207-217, 05/2015. tab, graf
Article in English | LILACS | ID: lil-748260

ABSTRACT

The practice of refrigerating raw milk at the farm has provided a selective advantage for psychrotrophic bacteria that produce heat-stable proteases and lipases causing severe quality problems to the dairy industry. In this work, a protease (AprX) and a lipase (LipM) produced by Pseudomonas fluorescens 041, a highly proteolytic and lipolytic strain isolated from raw milk obtained from a Brazilian farm, have been purified and characterized. Both enzymes were purified as recombinant proteins from Escherichia coli. The AprX metalloprotease exhibited activity in a broad temperature range, including refrigeration, with a maximum activity at 37 °C. It was active in a pH range of 4.0 to 9.0. This protease had maximum activity with the substrates casein and gelatin in the presence of Ca+2. The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from 7.0 to 10. It exhibited the highest activity, in the presence of Ca+2, on substrates with long-chain fatty acid residues. These results confirm the spoilage potential of strain 041 in milk due to, at least in part, these two enzymes. The work highlights the importance of studies of this kind with strains isolated in Brazil, which has a recent history on the implementation of the cold chain at the dairy farm.


Subject(s)
Animals , Lipase/metabolism , Milk/microbiology , Peptide Hydrolases/metabolism , Pseudomonas fluorescens/isolation & purification , Brazil , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Lipase/chemistry , Lipase/genetics , Lipase/isolation & purification , Peptide Hydrolases/chemistry , Peptide Hydrolases/genetics , Peptide Hydrolases/isolation & purification , Pseudomonas fluorescens/genetics , Refrigeration , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Temperature
7.
Recife; s.n; 2015. 86 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-983377

ABSTRACT

O exame parasitológico por Kato-Katz ainda é considerado o padrão ouro no diagnóstico da esquistossomose mansônica, entretanto, este apresenta baixa sensibilidade para utilização em inquéritos epidemiológicos. Além disso, as técnicas de diagnóstico imunológico, apresentam reações cruzadas com outros helmintos, protozoários e até bactérias como ocorre com a utilização dos extratos brutos do parasita. Nesse sentido, salientamos que Abath et al. identificaram um peptídeo de 15kDa denominado Sm15, que apresentou uma boa reatividade com soros de animais infectados pelo verme e, portanto, possui potencial para abordagens imunoprofiláticas e para testes diagnósticos. Neste estudo obtivemos o polipeptídio recombinante Sm15 em Escherichia coli e verificamos seu potencial para realização do diagnóstico a partir de amostras de soros de pacientes com diferentes manifestações clínicas da esquistossomose. Através de ELISA constatamos que o Sm15 apresentou maior reatividade frente a soros de pacientes esquistossomóticos, quando comparado ao extrato bruto SEA (P=0.0043). O Sm15 ainda demonstrou melhor desempenho ao apresentar maiores valores de sensibilidade, especificidade e área abaixo da curva ROC (P=0.0030). Além disso, o Sm15 foi capaz de diferenciar pacientes esquistossomóticos quanto à forma clínica, aguda ou crônica (P=0.0007). Os resultados obtidos neste estudo indicam, além de ratificar o potencial diagnóstico apresentado pelo polipeptídeo Sm15, que o mesmo poderá ser capaz de gerar uma alternativa de imunodiagnóstico de elevada acurácia, suprindo assim as lacunas existentes com relação aos testes parasitológicos e sorológicos atualmente disponíveis. Além disso, possibilitará o diagnostico precoce da esquistossomose, realização de inquéritos epidemiológicos em áreas de baixa endemicidade, impedindo assim a evolução da doença para formas clínicas de maior gravidade.


The parasitological examination by Kato-Katz still considered the gold standard in the diagnosis of schistosomiasis, however, it has low sensitivity for use in epidemiological surveys. Moreover, the techniques of immunological diagnosis, have cross-reactivity with other helminth, protozoa and even bacteria as occur with the use of crude parasite extracts. In this regard, we note that Abath and colleagues identified a 15kDa peptide termed SM15, which showed good reactivity with sera from animals infected by the worm, and therefore has potential immunoprophylactic and diagnostic testing approaches. In this study we obtained the recombinant polypeptide in Escherichia coli SM15 and check its potential for making the diagnosis from samples of patient sera with different clinical manifestations of schistosomiasis. By ELISA we found that the SM15 showed higher reactivity towards sera from schistosomiasis patients, when compared to the crude extract SEA (P = 0.0043). The SM15 also demonstrated better performance by presenting higher sensitivity, specificity, and area under the ROC curve (P = 0.0030). In addition, the SM15 was able to differentiate schistosomiasis patients about the clinical presentation, acute or chronic (P = 0.0007). The results of this study indicate not only ratifies the diagnostic potential presented by the SM15 polypeptide, that it may be able to generate an immunodiagnostic alternative high accuracy, thereby supplying the gaps in the parasitological and serological tests currently available. Also, it enables the early diagnosis of schistosomiasis, carrying out epidemiological surveys in low endemicity areas, thereby preventing disease progression to more severe clinical forms.


Subject(s)
Humans , Animals , Immunologic Tests/classification , Immunologic Tests/methods , Recombinant Proteins , Recombinant Proteins/isolation & purification , Schistosomiasis , Antigens, Helminth/genetics , Enzyme-Linked Immunosorbent Assay/methods , Helminth Proteins/genetics , Helminth Proteins/immunology , Schistosoma mansoni/genetics , Sensitivity and Specificity
8.
Braz. j. microbiol ; 45(2): 677-687, Apr.-June 2014. ilus, graf, tab
Article in English | LILACS | ID: lil-723134

ABSTRACT

A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca2+, Mg2+ and K+, while heavy metals (Fe3+ and Zn2+) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes.


Subject(s)
Enterobacter/enzymology , Lipase/isolation & purification , Lipase/metabolism , Amino Acid Sequence , Bacterial Typing Techniques , Base Sequence , Chromatography, Ion Exchange , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enzyme Stability , Enterobacter/classification , Enterobacter/genetics , Enterobacter/isolation & purification , Enzyme Activators/analysis , Enzyme Inhibitors/analysis , Escherichia coli/genetics , Gene Expression , Hydrogen-Ion Concentration , Iran , Lipase/chemistry , Lipase/genetics , Molecular Sequence Data , Molecular Weight , Open Reading Frames , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Soil Microbiology , Temperature
9.
Braz. j. microbiol ; 45(1): 351-358, 2014. ilus
Article in English | LILACS | ID: lil-709455

ABSTRACT

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2'-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 ºC, respectively . The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes.


Subject(s)
Laccase/genetics , Laccase/metabolism , Phytophthora/enzymology , Cloning, Molecular , Conserved Sequence , Enzyme Stability , Gene Expression , Hydrogen-Ion Concentration , Laccase/chemistry , Laccase/isolation & purification , Molecular Weight , Open Reading Frames , Protein Structure, Tertiary , Phytophthora/genetics , Pichia/genetics , Protein Sorting Signals/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Temperature
10.
Mem. Inst. Oswaldo Cruz ; 108(6): 671-678, set. 2013. tab, graf
Article in English | LILACS | ID: lil-685492

ABSTRACT

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.


Subject(s)
Animals , Female , Male , Gastrointestinal Tract/enzymology , Insect Vectors/parasitology , Phlebotomus/enzymology , Serine Proteinase Inhibitors/isolation & purification , CHO Cells , Cricetulus , Chymotrypsin/metabolism , Diptera/genetics , Gene Expression , Leishmaniasis/prevention & control , Life Cycle Stages/genetics , Psychodidae/parasitology , Regression Analysis , Reverse Transcriptase Polymerase Chain Reaction , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/metabolism , Thrombin/metabolism , Trypsin/metabolism
11.
Pesqui. vet. bras ; 33(1): 41-46, Jan. 2013. ilus, tab
Article in English | LILACS | ID: lil-668090

ABSTRACT

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.


Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 - que compartilha uma alta identidade com a gE do BoHV-5 - foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expressão em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.


Subject(s)
Animals , Cattle , Glycoproteins/isolation & purification , Herpesvirus 1, Bovine/isolation & purification , /isolation & purification , Recombinant Proteins/isolation & purification , Immunologic Tests/methods , Enzyme-Linked Immunosorbent Assay , Prokaryotic Cells , Vaccines, DNA
12.
Braz. j. microbiol ; 43(2): 544-551, Apr.-June 2012. ilus, graf
Article in English | LILACS | ID: lil-644469

ABSTRACT

Ostrich raising around the world have some key factors and farming profit depend largely on information and ability of farmers to rear these animals. Non fertilized eggs from ostriches are discharged in the reproduction season. Staphylococcus aureus and Escherichia coli are microorganisms involved in animal and human diseases. In order to optimize the use of sub products of ostrich raising, non fertilized eggs of four selected birds were utilized for development of polyclonal IgY antibodies. The birds were immunized (200ug/animal) with purified recombinant staphylococcal enterotoxin C (recSEC) and synthetic recRAP, both derived from S. aureus, and recBFPA and recEspB involved in E. coli pathogenicity, diluted in FCA injected in the braquial muscle. Two subsequent immunization steps with 21 days intervals were repeated in 0,85% saline in FIA. Blood and eggs samples were collected before and after immunization steps. Egg yolk immunoglobulins were purified by precipitation with 19% sodium sulfate and 20% ammonium sulphate methodologies. Purified IgY 50µL aliquots were incubated in 850µL BHI broth containing 50µL inoculums of five strains of S. aureus and five strains of E.coli during four hours at 37ºC. Growth inhibition was evaluated followed by photometry reading (DO550nm). Egg yolk IgY preparation from hiperimmunized birds contained antibodies that inhibited significantly (p<0,05) growth of strains tested. Potential use of ostrich IgY polyclonal antibodies as a diagnostic and therapeutic tool is proposed for diseased animals.


Subject(s)
Animals , Enterotoxins/isolation & purification , Escherichia coli/growth & development , Immunoglobulins/isolation & purification , Ovulation Inhibition , Recombinant Proteins/analysis , Recombinant Proteins/isolation & purification , Staphylococcus aureus/growth & development , Staphylococcus aureus/isolation & purification , Vaccination , Methods , Struthioniformes
13.
Article in English | WPRIM | ID: wpr-197697

ABSTRACT

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh(s)) and a 33 kDa beta-domain (Tsh(beta)). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh(s) (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh(beta) (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37degrees C or 42degrees C. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26degrees C, 37degrees C, or 42degrees C, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37degrees C in mucin agar, and it was not detected when the bacteria were grown at 26degrees C in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.


Subject(s)
Adhesins, Escherichia coli/metabolism , Brazil , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Hemagglutination , Mucins/metabolism , Protein Transport , Recombinant Proteins/isolation & purification
14.
São Paulo; s.n; 16 dez. 2008. 148[15] p. ilus, graf, tab.
Thesis in Portuguese | LILACS | ID: lil-508068

ABSTRACT

Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As Iisozimas digestivas de insetos são do tipo "c" e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 — AAQ20048 e lisozima 2 — AAQ20047) da larva de Musca domestica (mosca — Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de p’K IND.a’s do nucleófilo e do doador de prótons...


Subject(s)
Animals , Digestion/physiology , Diptera/enzymology , Diptera/genetics , Enzymes , Molecular Biology , Muramidase/physiology , Muramidase/isolation & purification , Pichia/enzymology , Pichia/isolation & purification , Recombinant Proteins/isolation & purification , Cell Culture Techniques , Electrophoresis, Agar Gel , Polymerase Chain Reaction
15.
Indian J Biochem Biophys ; 2007 Aug; 44(4): 204-8
Article in English | IMSEAR | ID: sea-27993

ABSTRACT

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.


Subject(s)
Aeromonas hydrophila/metabolism , Bacterial Toxins/immunology , Chromatography, Affinity , Cloning, Molecular/methods , Escherichia coli/genetics , Pore Forming Cytotoxic Proteins/immunology , Protein Sorting Signals/genetics , Recombinant Proteins/isolation & purification
16.
Article in English | WPRIM | ID: wpr-634171

ABSTRACT

To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.


Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Plasmids/biosynthesis , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , SARS Virus/chemistry , SARS Virus/genetics , Viral Vaccines/biosynthesis
17.
São Paulo; s.n; 2003. 95 p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: lil-344581

ABSTRACT

Bordetella pertussis, o agente etiológico da coqueluche ou tosse comprida, que estabelece a infecção através da fixação bacteriana no epitélio do rato respiratório superior. Os principais mediadores de adesão da bactéria são a toxina pertússica (PT) e a hemaglutinina filamentosa (FHA). A FHA é a adesina majoritária e contém pelo menos 4 domínios: porção N-terminal, domínio de reconhecimento de carboidratos (CRD) (`FHA IND. 1141-1279ï), trinca de aminoácidos Arginina-Glicina-Ácido aspártico (RGD) (`FHA IND. 1097-1099ï) e o sítio de ligação a heparina (domínio Hep) (`FHA IND. 442-863ï). Neste trabalho, foi realizado a amplificação de duas regiões do domínio de ligação à heparina, as regiões MAL80 (`FHA IND. 299-873ï) e HEP (`FHA IND. 430-873ï)...


Subject(s)
Animals , Mice , Bordetella pertussis , Hemagglutination , Heparin , Recombinant Proteins/isolation & purification , Antibody Formation , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Polymerase Chain Reaction/methods
18.
Indian J Biochem Biophys ; 2001 Oct; 38(5): 303-8
Article in English | IMSEAR | ID: sea-28042

ABSTRACT

The type II restriction endonuclease, Bam HI, has been overexpressed in E. coli by cloning the Bam HI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of Bam HI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK14. Our overexpressing clone, pAABRw in BL21 cells in presence of Bam HI methylase in pMAP6 following induction with IPTG yields about 9.2 x 10(6) units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH139 cells even in the absence of T7 polymerase and cognate Bam HI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.


Subject(s)
Binding Sites , Cell Division , Cells, Cultured , DNA-Directed RNA Polymerases/metabolism , Deoxyribonuclease BamHI/genetics , Mutation , Plasmids , Recombinant Proteins/isolation & purification , Viral Proteins
19.
Mem. Inst. Oswaldo Cruz ; 96(suppl): 131-135, Sept. 2001. ilus, tab
Article in English | SES-SP, LILACS, SES-SP | ID: lil-295892

ABSTRACT

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system


Subject(s)
Animals , Female , Mice , Schistosoma mansoni/immunology , Recombinant Proteins , Antibodies, Helminth/physiology , Helminth Proteins/physiology , Plasmids , Recombinant Proteins/isolation & purification , Carrier Proteins , Helminth Proteins/isolation & purification , Blotting, Western , Amino Acid Sequence , Vaccination , DNA, Complementary , Models, Animal , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Fatty Acids
20.
Biomédica (Bogotá) ; 20(1): 10-7, mar. 2000. ilus, tab
Article in Spanish | LILACS | ID: lil-278057

ABSTRACT

El presente trabajo describe la producción de la proteína recombinante JAK2, empleando un sistema de vectores de expresión baculovirales. La infección de células Sf9, derivadas del tejido de ovario de Spodoptera frugiperda, permitió establecer las condiciones óptimas de dilución del virus en 10 µl/ml de medio y de tiempo de recolección en 72 horas post-infección. Se recolectaron y ultrafiltraron extractos correspondientes a la infección de 5 x 10 a la 7 células, aproximadamente, y se sometieron a cromatografía de intercambio iónico (DEAE-Sephacel), lográndose purificar parcialmente la proteína JAK2 por medio de un gradiente continuo de fuerza iónica (NaCl 10-500 mM). La presencia de JAK2 se verificó por Western blot, empleando un anticuerpo policlonal producido en conejo contra los residuos 758-776 de JAK2 de ratón. Los resultados indicaron que la proteína obtenida por este sistema de expresión se sintetiza en forma fosforilada/activada


Subject(s)
Mice , Recombinant Proteins/isolation & purification , Receptors, Somatotropin , Baculoviridae
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