A new vector for heterologous gene expression in Escherichia coli with increased stability in the absence of antibiotic.

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.

A new vector for heterologous gene expression in Escherichia coli with increased stability in the absence of antibiotic

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.

A new vector for heterologous gene expression in Escherichia coli with increased stability in the absence of antibiotic

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.

Cross neutralization of coral snake venoms by commercial Australian snake antivenoms.

CONTEXT: Although rare, coral snake envenomation is a serious health threat in Brazil, because of the highly neurotoxic venom and the scarcely available antivenom. The major bottleneck for antivenom production is the low availability of venom. Furthermore, the available serum is not effective against all coral snake species found in Brazil. An alternative to circumvent the lack of venom for serum production and the restricted protection of the actually available antivenom would be of great value. We compared the Brazilian coral snake and mono and polyvalent Australian antivenoms in terms of reactivity and protection. METHODS: The immunoreactivity of venoms from 9 coral snakes species were assayed by ELISA and western blot using the Brazilian Micrurus and the Australian pentavalent as well as monovalent anti-Notechis, Oxyuranus and Pseudechis antivenoms. Neutralization assays were performed in mice, using 3 LD50 of the venoms, incubated for 30 minutes with 100 µL of antivenom/animal. DISCUSSION: All the venoms reacted against the autologous and heterologous antivenoms. Nevertheless, the neutralization assays showed that the coral snake antivenom was only effective against M. corallinus, M. frontalis, M. fulvius, M. nigrocinctus and M. pyrrhocryptus venoms. On the other hand, the Australian pentavalent antivenom neutralized all venoms except the one from M. spixii. A combination of anti-Oxyuranus and Pseudechis monovalent sera, extended the protection to M. altirostris and, partially, to M. ibiboboca. By adding Notechis antivenom to this mixture, we obtained full protection against M. ibiboboca and partial neutralization against M. lemniscatus venoms. CONCLUSIONS: Our findings confirm the limited effectiveness of the Brazilian coral snake antivenom and indicate that antivenoms made from Australian snakes venoms are an effective alternative for coral snake bites in South America and also in the United States were coral snake antivenom production has been discontinued.

Cross neutralization of coral snake venoms by commercial Australian snake antivenoms

Context: Although rare, coral snake envenomation is a serious health threat in Brazil, because of the highly neurotoxic venom and the scarcely available antivenom. The major bottleneck for antivenom production is the low availability of venom. Furthermore, the available serum is not effective against all coral snake species found in Brazil. An alternative to circumvent the lack of venom for serum production and the restricted protection of the actually available antivenom would be of great value. We compared the Brazilian coral snake and mono and polyvalent Australian antivenoms in terms of reactivity and protection. Methods: The immunoreactivity of venoms from 9 coral snakes species were assayed by ELISA and western blot using the Brazilian Micrurus and the Australian pentavalent as well as monovalent antiNotechis, Oxyuranus and Pseudechis antivenoms. Neutralization assays were performed in mice, using 3 LD50 of the venoms, incubated for 30 minutes with 100 mu L of antivenom/animal. Discussion: All the venoms reacted against the autologous and heterologous antivenoms. Nevertheless, the neutralization assays showed that the coral snake antivenom was only effective against M. corallinus, M. frontalis, M. fulvius, M. nigrocinctus and M. pyrrhocryptus venoms. On the other hand, the Australian pentavalent antivenom neutralized all venoms except the one from M. spixii. A combination of anti-Oxyuranus and Pseudechis monovalent sera, extended the protection to M. altirostris and, partially, to M. ibiboboca. By adding Notechis antivenom to this mixture, we obtained full protection against M. ibiboboca and partial neutralization against M. lemniscatus venoms. Conclusions: Our findings confirm the limited effectiveness of the Brazilian coral snake antivenom and indicate that antivenoms made from Australian snakes venoms are an effective alternative for coral snake bites in South America and also in the United States were coral snake antivenom production has been discontinued.

A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom.

BACKGROUND: Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. METHODS AND FINDINGS: In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. CONCLUSION: Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.

A Genetic Fusion between Sm14 and CTB does not Reduce Schistosoma mansoni Worm Burden on IntranasallyImmunized BALB/c Mice

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.

Desenvolvimento e caracterização de uma vacina antiesquistossomose composta pela proteína Sm14 de Schistosoma mansoni utilizando a subunidade B da toxina colérica (CTB) como adjuvante / Development and characterization of an antischistosomiasis vaccine composed by Sm14 protein, from Schistosoma mansoni, using the B subunit of cholera toxin (CTB) as adjuvant

Introdução: o desenvolvimento de uma vacina contra a esquistossomose será um importante avanço no controle desta doença crônica e muitas vezes debilitante, afetando milhões de pessoas em todo o mundo. Neste trabalho, descrevemos o uso da subunidade B da toxina colérica (CTB) geneticamente fusionada com Sm14 - uma proteína ligadora de ácidos graxos de Schistosoma mansoni - como uma tentativa de desenvolver uma vacina antiesquistossomose. Métodos: proteínas recombinantes foram expressas em um sistema procariótico, purificadas por diferentes métodos cromatográficos e caracterizadas tanto por métodos imunoquímicos como por métodos espectroscópicos. Experimentos de imunização foram realizados em camundongos fêmeas, da linhagem BALB/c e a eficácia da vacina determinada através da análise da carga parasitária após o desafio com cercárias de S. mansoni e através da análise histopatológica das reações granulomatosas ao redor dos ovos aprisionados no tecido hepático dos camundongos. Resultados: a administração subcutânea de Sm14 reduziu em 27 por cento a carga parasitária nos animais vacinados. Por outro lado, a vacinação intranasal apenas demonstrou uma redução estatisticamente significativa quando CTB esteve presente na formulação...

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp

Leptospirosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-Å-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL3221-272). The LipL3221-272 monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL3221-272 is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL3221-272. Ca2+ binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca2+ as no structural or stability perturbations were observed for Mg2+, Zn2+, or Cu2+. Careful examination of the crystallographic structure suggests the locations of putative regions that could mediate Ca2+ binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and laminin.