RESUMO
Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244⯵g/mL of rSm14, 181⯵g/mL and 392⯵g/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147⯵g/mL of rSm14 and 162⯵g/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli, even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins.
Assuntos
Antibacterianos , Escherichia coli/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Vetores Genéticos/química , Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas de Helminto/metabolismo , Plasmídeos/química , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Transporte de Ácido Graxo/química , Proteínas de Transporte de Ácido Graxo/genética , Vetores Genéticos/genética , Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Using a cDNA library made from Lonomia obliqua caterpillar bristles, we identified a transcript with a 603 bp open reading frame. The deduced protein corresponds to Lopap, a prothrombin activator previously isolated by our group from the bristles of this species. The mature protein is composed by 185 amino acids and shares similarity with members of the lipocalin family. The cDNA encoding the mature form was amplified by PCR, subcloned into pAE vector and used to transform Escherichia coli BL21(DE3) cells. As for the native Lopap, the recombinant fusion protein shows enzymatic activity, promotes prothrombin hydrolysis, generates fragments similar to prethrombin-2 and fragment 1.2 as intermediates, and generates thrombin as the final product. In addition, structural bioinformatics studies indicated several interesting molecular features, including the residues that could be responsible for Lopap's serine protease-like activity and the role of calcium binding in this context. Such catalytic activity has never been found in other members of the lipocalin family. This is the first report describing the recombinant production and biochemical characterization of a Lonomia obliqua lipocalin, as well as the structural features that could be responsible for its serine protease-like catalytic activity.
Assuntos
Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Dicroísmo Circular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Fator X/metabolismo , Humanos , Hidrólise , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Lactoglobulinas/química , Lepidópteros , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Protrombina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/classificação , Serina Endopeptidases/genética , Relação Estrutura-AtividadeRESUMO
Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244?µg/mL of rSm14, 181?µg/mL and 392?µg/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147?µg/mL of rSm14 and 162?µg/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli, even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins.
RESUMO
Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244?µg/mL of rSm14, 181?µg/mL and 392?µg/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147?µg/mL of rSm14 and 162?µg/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli, even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins.
RESUMO
The exosome is a conserved eukaryotic enzymatic complex that plays an essential role in many pathways of RNA processing and degradation. Here, we describe the structural characterization of the predicted archaeal exosome in solution using small angle x-ray scattering. The structure model calculated from the small angle x-ray scattering pattern provides an indication of the existence of a disk-shaped structure, corresponding to the "RNases PH ring" complex formed by the proteins aRrp41 and aRrp42. The RNases PH ring complex corresponds to the core of the exosome, binds RNA, and has phosphorolytic and polymerization activities. Three additional molecules of the RNA-binding protein aRrp4 are attached to the core as extended and flexible arms that may direct the substrates to the active sites of the exosome. In the presence of aRrp4, the activity of the core complex is enhanced, suggesting a regulatory role for this protein. The results shown here also indicate the participation of the exosome in RNA metabolism in Archaea, as was established in Eukarya.
Assuntos
Pyrococcus/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA Arqueal/química , RNA Arqueal/fisiologia , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/fisiologia , Cromatografia em Gel , Ensaio de Desvio de Mobilidade Eletroforética , Exorribonucleases/química , Exorribonucleases/metabolismo , Modelos Moleculares , Ligação Proteica , Pyrococcus/química , Pyrococcus/enzimologia , Espalhamento de Radiação , Soluções , Difração de Raios X , Raios XRESUMO
Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.
Assuntos
Toxina da Cólera/genética , Toxina da Cólera/isolamento & purificação , Genes Bacterianos/genética , Histidina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Glândulas Suprarrenais/citologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Linhagem Celular , Toxina da Cólera/biossíntese , Toxina da Cólera/química , Códon/genética , DNA Recombinante/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Histidina/genética , Dados de Sequência Molecular , Subunidades Proteicas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Vibrio cholerae/químicaRESUMO
The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His(6)-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant beta-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14-M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20>rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.
Assuntos
Proteínas de Transporte/genética , Proteínas de Helminto/genética , Proteínas de Membrana Transportadoras , Proteínas de Neoplasias , Polimorfismo Genético , Schistosoma mansoni/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Brasil , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dicroísmo Circular , Primers do DNA , Proteínas de Transporte de Ácido Graxo , Proteínas de Ligação a Ácido Graxo , Feminino , Geografia , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Íntrons , Masculino , Dados de Sequência Molecular , Família Multigênica , Mutação de Sentido Incorreto , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
Developing a vaccine against schistosomiasis would be an important advance on the control of this chronic and debilitating disease that afflicts millions of people worldwide. Herein we describe the use of the non-toxic B subunit of cholera toxin (CTB) genetically fused to Sm14 - a fatty-acid binding protein from Schistosoma mansoni - as an attempt to elicit a mucosal immune response against the lung stage of this parasite by intranasal immunization. Recombinant proteins were expressed on a prokaryotic system, purified by affinity chromatography and both immunochemically and spectroscopically characterized. Intranasal immunization experiments were performed on BALB/c mice and vaccine efficacy was assessed analyzing the worm-burden after challenge infection with S. mansoni cercariae. The results demonstrate that Sm14 itself was not able to reduce the worm burden on intranasally vaccinated animals. The presence of CTB either in intranasal coadministration with or genetically fused to Sm14 did not significantly improve the protective response of Sm14 as a worm burden reduction of only 20% could be observed. In addition to that, however, CTB demonstrated a clear anti inflammatory effect on the liver of immunized mice, which displayed hepatic granulomas around trapped eggs 15% smaller than control groups, indicating that CTB displays an immunomodulatory effect on the inflammatory responses induced by the parasite egg toxins.
Assuntos
Humanos , Esquistossomose/imunologia , VacinasRESUMO
Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending onthe antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, wesynthesized a versatile gene coding a 6XHis-tagged CTB (359 bp). The sequence was designed according to codon usage of Escherichiacoli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in whichthe polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, andexpressed in E. coli in an insoluble form, reaching levels about 13mg of purified active pentameric rCTB per liter of induced culture.Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodiesagainst the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxinactivity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functionalCTB through Ni2þ-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained fromVibrio cholerae cultures.
Assuntos
Masculino , Feminino , Humanos , Clonagem Molecular , Células Cultivadas , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
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
Assuntos
Animais , Feminino , Camundongos , Schistosoma mansoni/imunologia , Proteínas Recombinantes , Anticorpos Anti-Helmínticos/fisiologia , Proteínas de Helminto/fisiologia , Plasmídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas de Transporte , Proteínas de Helminto/isolamento & purificação , Western Blotting , Sequência de Aminoácidos , Vacinação , DNA Complementar , Modelos Animais , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Ácidos GraxosRESUMO
A esquistossomose é a mais importante das doenças helmínticas humanas em termos de morbidez e mortalidade. A proteína Sm14 de Schistosoma mansoni, que pertence à família de proteínas ligadoras de ácidos graxos (fatty acid-binding proteins, FABPs) (Moser et al., 1991), mostrou um bom nível de proteção (65 por cento) contra a esquistossomose em animais experimentais (Tendler et al., 1996). No presente trabalho foram desenvolvidos sistemas de expressão que possibilitará a produção da proteína Sm14 em larga escala em E. coli. Com o intuito de conhecer a estrutura do gene da proteína Sm14, foi clonado um fragmento de DNA genômico de S. mansoni que contém a seqüência codificante da proteína Sm14...