Expressão e caracterização da glicoproteína D do herpesvírus equídeo 1 em Pichia pastoris / Expression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris

O herpesvírus equídeo 1 (EHV-1) apresenta distribuição mundial e causa graves prejuízos à equideocultura. É agente de surtos de doença respiratória, reprodutiva e neurológica, em equídeos jovens e adultos. A glicoproteína D (gD) do envelope viral é essencial para ligação e penetração em células permissivas e direcionamento do sistema imunológico do hospedeiro, induz respostas imunes humorais e celulares, sendo um antígeno apropriado para ser utilizado em vacinas e imunodiagnóstico. O objetivo deste trabalho foi expressar e caracterizar a gD do EHV-1 em Pichia pastoris para posterior utilização como antígeno em técnicas de imunodiagnóstico e formulação de vacinas recombinantes. Uma sequência de DNA que codifica uma forma truncada da gDEHV-1 foi clonada no vetor pPICZαA de expressão em P. pastoris. Obteve-se uma proteína de ~41 kDa, como esperado. A proteína apresentou glicosilação entre 4 kDa e 16 kDa, demonstrada por deglicosilação enzimática. A proteína recombinante foi caracterizada antigenicamente e imunogenicamente por Western blot, utilizando-se anticorpos policlonais equinos anti-EHV-1, e por ELISA indireto em modelo murino, demonstrando que a gD recombinante manteve epítopos similares aos da proteína nativa. Esses resultados sugerem que a gDEHV-1 é um antígeno promissor para uso como imunobiológico no controle do EHV-1.(AU)

Equine herpesvirus 1 (EHV-1) has a worldwide distribution and causes serious damage to horse breeding. It is an agent of respiratory, reproductive and neurological disease outbreaks in young and adult equids. Viral envelope glycoprotein D (gD) is essential for binding and penetration into permissive cells and targeting the host immune system, inducing humoral and cellular immune responses, and is an appropriate antigen for use in vaccines and immunodiagnostics. The objective of this work was to express in Pichia pastoris and to characterize EHV-1 gD for later use as an antigen in immunodiagnostic techniques and formulation of recombinant vaccines. A DNA sequence encoding a truncated form of gDEHV-1 has been cloned into the P. pastoris expression vector pPICZαA. A protein of ~41 kDa was obtained as expected. The protein presented glycosylation between 4 kDa and 16 kDa, demonstrated by enzymatic deglycosylation. The recombinant protein was antigenically and immunogenically characterized by Western blot using equine polyclonal anti-EHV-1 antibodies, and by indirect ELISA in a murine model, demonstrating that the recombinant gD maintained epitopes similar to those of the native protein. These results suggest that gDEHV-1 is a promising antigen for use as an immunobiological in the control of EHV-1.(AU)

Expressão e caracterização da glicoproteína D do herpesvírus equídeo 1 em Pichia pastoris / Expression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris

O herpesvírus equídeo 1 (EHV-1) apresenta distribuição mundial e causa graves prejuízos à equideocultura. É agente de surtos de doença respiratória, reprodutiva e neurológica, em equídeos jovens e adultos. A glicoproteína D (gD) do envelope viral é essencial para ligação e penetração em células permissivas e direcionamento do sistema imunológico do hospedeiro, induz respostas imunes humorais e celulares, sendo um antígeno apropriado para ser utilizado em vacinas e imunodiagnóstico. O objetivo deste trabalho foi expressar e caracterizar a gD do EHV-1 em Pichia pastoris para posterior utilização como antígeno em técnicas de imunodiagnóstico e formulação de vacinas recombinantes. Uma sequência de DNA que codifica uma forma truncada da gDEHV-1 foi clonada no vetor pPICZαA de expressão em P. pastoris. Obteve-se uma proteína de ~41 kDa, como esperado. A proteína apresentou glicosilação entre 4 kDa e 16 kDa, demonstrada por deglicosilação enzimática. A proteína recombinante foi caracterizada antigenicamente e imunogenicamente por Western blot, utilizando-se anticorpos policlonais equinos anti-EHV-1, e por ELISA indireto em modelo murino, demonstrando que a gD recombinante manteve epítopos similares aos da proteína nativa. Esses resultados sugerem que a gDEHV-1 é um antígeno promissor para uso como imunobiológico no controle do EHV-1.(AU)

Equine herpesvirus 1 (EHV-1) has a worldwide distribution and causes serious damage to horse breeding. It is an agent of respiratory, reproductive and neurological disease outbreaks in young and adult equids. Viral envelope glycoprotein D (gD) is essential for binding and penetration into permissive cells and targeting the host immune system, inducing humoral and cellular immune responses, and is an appropriate antigen for use in vaccines and immunodiagnostics. The objective of this work was to express in Pichia pastoris and to characterize EHV-1 gD for later use as an antigen in immunodiagnostic techniques and formulation of recombinant vaccines. A DNA sequence encoding a truncated form of gDEHV-1 has been cloned into the P. pastoris expression vector pPICZαA. A protein of ~41 kDa was obtained as expected. The protein presented glycosylation between 4 kDa and 16 kDa, demonstrated by enzymatic deglycosylation. The recombinant protein was antigenically and immunogenically characterized by Western blot using equine polyclonal anti-EHV-1 antibodies, and by indirect ELISA in a murine model, demonstrating that the recombinant gD maintained epitopes similar to those of the native protein. These results suggest that gDEHV-1 is a promising antigen for use as an immunobiological in the control of EHV-1.(AU)

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1. Methods: Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins. Results: When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis Conclusion: These results suggest that the recombinant enzyme has no potential as a thrombolytic agent but can be applied in the prevention of thrombus formation in some pathological processes and as molecular tools in studies related to hemostasis.(AU)

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

Background:The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1.Methods:Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins.Results:When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis...(AU)

Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast

Abstract Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.

Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast

Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.(AU)

Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris

The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 ºC + 1%M provided the most stable viability. By contrast, the temperature of 18 ºC + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.(AU)

Dynamics of humoral immune response in pregnant mares and foals vaccinated with Theileria equi recombinant EMA-2 / Dinâmica da resposta imune humoral em éguas gestantes e potros vacinados com EMA-2 recombinante de Theileria equi

Theileria equi is an infectious hemoprotozoan agent of equine piroplasmosis, a disease that has severe economic and sanitary impact internationally. In addition to its common clinical features, piroplasmosis can cause gestational losses and neonatal damage, which makes neonates susceptible to this disease. The aim of this study was to evaluate the dynamics of humoral immune response to recombinant EMA-2 of T. equi in pregnant mares and foals, as well as the transfer of vaccine antibodies through the colostrum ingested by sucking foals. For vaccine production, the EMA-2 expression gene was cloned and expressed in the yeast species, Pichia pastoris. Thirty-six horses were used, of which 18 were pregnant mares and 18 were foals. The mares were divided into control and vaccinated groups, and the vaccinated group received three doses of rEMA-2 every 21 days starting at 300 days of gestation. Foals from vaccinated and control groups were evaluated until the sixth month of life. The production of antibodies by foals on the rEMA-2 vaccination schedule was also evaluated from the second month of life. Foals in the vaccinated group had received three doses of the vaccine every 21 days. The method used to evaluate serum and colostrum samples was indirect ELISA, and plates were sensitized with the rEMA-2 protein. At the end of the vaccination schedule, vaccinated mares showed a 2.3-fold increase in antibody levels when compared to baseline values. The colostrum of vaccinated mares presented antibody levels of 1.0432±0.33. Foals delivered by vaccinated mares presented levels of antibodies greater than those of foals delivered by control mares after their first time sucking (at about twelve hours after birth). Foals vaccinated in the second month of life showed an 8.3-fold increase in antibody levels when compared to baseline values. The vaccination schedule with rEMA-2 was able to stimulate humoral immunity in pregnant mares...(AU)

Theileria equi é um hemoprotozoário, agente da piroplasmose equina, doença de impacto sanitário e econômico internacional. Em éguas gestantes além da doença clínica, podem ocorrer abortos e danos ao neonato, caracterizando grande susceptibilidade à doença no período neonatal. O objetivo deste estudo foi avaliar a dinâmica da resposta imune humoral à EMA-2 recombinante de T. equi em éguas gestantes e potros, bem como a transferência de anticorpos vacinais no colostro. Foram utilizados 36 equinos, sendo 18 éguas gestantes e 18 potros. As éguas foram divididas em grupo controle e vacinado, que receberam rEMA-2 a partir dos 300 dias de gestação em três doses com intervalos de 21 dias. Para produção da vacina, o gene de expressão de EMA-2 foi clonado e a proteína expressa em Pichia pastoris. Os potros provenientes de éguas dos grupos vacinado e controle foram avaliados até o 6º mês de vida. Avaliou-se também a produção de anticorpos em potros submetidos ao esquema vacinal com rEMA-2 a partir do 2º mês de vida, que receberam três doses da vacina em intervalos de 21 dias. O método escolhido para a avaliação das amostras de soro e colostro foi ELISA indireto, com sensibilização pela proteína rEMA-2. Nas éguas gestantes vacinadas com rEMA-2 ocorreu o incremento de 2,3 vezes o valor basal ao final do esquema vacinal. O colostro de éguas vacinadas apresentou título médio de anticorpos de 1,0432±0,33, e potros provenientes de éguas vacinadas apresentaram média maior que os provenientes de éguas controle após a primeira mamada (12 horas). Os potros que passaram por esquema vacinal a partir do 2º mês de vida obtiveram incremento de 8,3 vezes o valor basal de anticorpos. O esquema vacinal com rEMA-2 foi capaz de estimular a imunidade humoral em éguas gestantes. Éguas gestantes vacinadas concentraram imunoglobulinas vacinais no colostro, e os potros provenientes destas obtiveram incremento nos níveis séricos de anticorpos vacinais após a primeira mamada.(AU)

Dynamics of humoral immune response in pregnant mares and foals vaccinated with Theileria equi recombinant EMA-2

ABSTRACT: Theileria equi is an infectious hemoprotozoan agent of equine piroplasmosis, a disease that has severe economic and sanitary impact internationally. In addition to its common clinical features, piroplasmosis can cause gestational losses and neonatal damage, which makes neonates susceptible to this disease. The aim of this study was to evaluate the dynamics of humoral immune response to recombinant EMA-2 of T. equi in pregnant mares and foals, as well as the transfer of vaccine antibodies through the colostrum ingested by sucking foals. For vaccine production, the EMA-2 expression gene was cloned and expressed in the yeast species, Pichia pastoris. Thirty-six horses were used, of which 18 were pregnant mares and 18 were foals. The mares were divided into control and vaccinated groups, and the vaccinated group received three doses of rEMA-2 every 21 days starting at 300 days of gestation. Foals from vaccinated and control groups were evaluated until the sixth month of life. The production of antibodies by foals on the rEMA-2 vaccination schedule was also evaluated from the second month of life. Foals in the vaccinated group had received three doses of the vaccine every 21 days. The method used to evaluate serum and colostrum samples was indirect ELISA, and plates were sensitized with the rEMA-2 protein. At the end of the vaccination schedule, vaccinated mares showed a 2.3-fold increase in antibody levels when compared to baseline values. The colostrum of vaccinated mares presented antibody levels of 1.0432±0.33. Foals delivered by vaccinated mares presented levels of antibodies greater than those of foals delivered by control mares after their first time sucking (at about twelve hours after birth). Foals vaccinated in the second month of life showed an 8.3-fold increase in antibody levels when compared to baseline values. The vaccination schedule with rEMA-2 was able to stimulate humoral immunity in pregnant mares. Vaccine immunoglobins were concentrated in the colostrum of vaccinated mares and foals delivered by these mares showed an increase in serum levels of vaccine antibodies after the first-time sucking.

RESUMO: Theileria equi é um hemoprotozoário, agente da piroplasmose equina, doença de impacto sanitário e econômico internacional. Em éguas gestantes além da doença clínica, podem ocorrer abortos e danos ao neonato, caracterizando grande susceptibilidade à doença no período neonatal. O objetivo deste estudo foi avaliar a dinâmica da resposta imune humoral à EMA-2 recombinante de T. equi em éguas gestantes e potros, bem como a transferência de anticorpos vacinais no colostro. Foram utilizados 36 equinos, sendo 18 éguas gestantes e 18 potros. As éguas foram divididas em grupo controle e vacinado, que receberam rEMA-2 a partir dos 300 dias de gestação em três doses com intervalos de 21 dias. Para produção da vacina, o gene de expressão de EMA-2 foi clonado e a proteína expressa em Pichia pastoris. Os potros provenientes de éguas dos grupos vacinado e controle foram avaliados até o 6º mês de vida. Avaliou-se também a produção de anticorpos em potros submetidos ao esquema vacinal com rEMA-2 a partir do 2º mês de vida, que receberam três doses da vacina em intervalos de 21 dias. O método escolhido para a avaliação das amostras de soro e colostro foi ELISA indireto, com sensibilização pela proteína rEMA-2. Nas éguas gestantes vacinadas com rEMA-2 ocorreu o incremento de 2,3 vezes o valor basal ao final do esquema vacinal. O colostro de éguas vacinadas apresentou título médio de anticorpos de 1,0432±0,33, e potros provenientes de éguas vacinadas apresentaram média maior que os provenientes de éguas controle após a primeira mamada (12 horas). Os potros que passaram por esquema vacinal a partir do 2º mês de vida obtiveram incremento de 8,3 vezes o valor basal de anticorpos. O esquema vacinal com rEMA-2 foi capaz de estimular a imunidade humoral em éguas gestantes. Éguas gestantes vacinadas concentraram imunoglobulinas vacinais no colostro, e os potros provenientes destas obtiveram incremento nos níveis séricos de anticorpos vacinais após a primeira mamada.

Dynamics of humoral immune response in pregnant mares and foals vaccinated with Theileria equi recombinant EMA-2 / Dinâmica da resposta imune humoral em éguas gestantes e potros vacinados com EMA-2 recombinante de Theileria equi

Theileria equi is an infectious hemoprotozoan agent of equine piroplasmosis, a disease that has severe economic and sanitary impact internationally. In addition to its common clinical features, piroplasmosis can cause gestational losses and neonatal damage, which makes neonates susceptible to this disease. The aim of this study was to evaluate the dynamics of humoral immune response to recombinant EMA-2 of T. equi in pregnant mares and foals, as well as the transfer of vaccine antibodies through the colostrum ingested by sucking foals. For vaccine production, the EMA-2 expression gene was cloned and expressed in the yeast species, Pichia pastoris. Thirty-six horses were used, of which 18 were pregnant mares and 18 were foals. The mares were divided into control and vaccinated groups, and the vaccinated group received three doses of rEMA-2 every 21 days starting at 300 days of gestation. Foals from vaccinated and control groups were evaluated until the sixth month of life. The production of antibodies by foals on the rEMA-2 vaccination schedule was also evaluated from the second month of life. Foals in the vaccinated group had received three doses of the vaccine every 21 days. The method used to evaluate serum and colostrum samples was indirect ELISA, and plates were sensitized with the rEMA-2 protein. At the end of the vaccination schedule, vaccinated mares showed a 2.3-fold increase in antibody levels when compared to baseline values. The colostrum of vaccinated mares presented antibody levels of 1.0432±0.33. Foals delivered by vaccinated mares presented levels of antibodies greater than those of foals delivered by control mares after their first time sucking (at about twelve hours after birth). Foals vaccinated in the second month of life showed an 8.3-fold increase in antibody levels when compared to baseline values. The vaccination schedule with rEMA-2 was able to stimulate humoral immunity in pregnant mares. Vaccine immunoglobins were concentrated in the colostrum of vaccinated mares and foals delivered by these mares showed an increase in serum levels of vaccine antibodies after the first-time sucking.(AU)

Theileria equi é um hemoprotozoário, agente da piroplasmose equina, doença de impacto sanitário e econômico internacional. Em éguas gestantes além da doença clínica, podem ocorrer abortos e danos ao neonato, caracterizando grande susceptibilidade à doença no período neonatal. O objetivo deste estudo foi avaliar a dinâmica da resposta imune humoral à EMA-2 recombinante de T. equi em éguas gestantes e potros, bem como a transferência de anticorpos vacinais no colostro. Foram utilizados 36 equinos, sendo 18 éguas gestantes e 18 potros. As éguas foram divididas em grupo controle e vacinado, que receberam rEMA-2 a partir dos 300 dias de gestação em três doses com intervalos de 21 dias. Para produção da vacina, o gene de expressão de EMA-2 foi clonado e a proteína expressa em Pichia pastoris. Os potros provenientes de éguas dos grupos vacinado e controle foram avaliados até o 6º mês de vida. Avaliou-se também a produção de anticorpos em potros submetidos ao esquema vacinal com rEMA-2 a partir do 2º mês de vida, que receberam três doses da vacina em intervalos de 21 dias. O método escolhido para a avaliação das amostras de soro e colostro foi ELISA indireto, com sensibilização pela proteína rEMA-2. Nas éguas gestantes vacinadas com rEMA-2 ocorreu o incremento de 2,3 vezes o valor basal ao final do esquema vacinal. O colostro de éguas vacinadas apresentou título médio de anticorpos de 1,0432±0,33, e potros provenientes de éguas vacinadas apresentaram média maior que os provenientes de éguas controle após a primeira mamada (12 horas). Os potros que passaram por esquema vacinal a partir do 2º mês de vida obtiveram incremento de 8,3 vezes o valor basal de anticorpos. O esquema vacinal com rEMA-2 foi capaz de estimular a imunidade humoral em éguas gestantes. Éguas gestantes vacinadas concentraram imunoglobulinas vacinais no colostro, e os potros provenientes destas obtiveram incremento nos níveis séricos de anticorpos vacinais após a primeira mamada.(AU)

Production of recombinant human epidermal growth factor in Pichia pastoris

This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.(AU)

Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source

Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8h.(AU)

Potencial terapêutico das proteínas imunorregulatórias recombinantes: produção heteróloga, caracterização e avaliação da atividade biológica da Cistatina-B da piranha vermelha Pygocentrus nattereri

A aquicultura é o setor de maior crescimento entre as indústrias de produção animal. Paralelo a esse importante crescimento ocorre o surgimento de doenças infecciosas, as quais podem causar significativas perdas nos estoques e problemas com o bem-estar animal, sendo o maior obstáculo para avanços na produção aquícola. Para enfrentar essa problemática é necessário associar boas técnicas de manejo ao desenvolvimento de novas tecnologias. Áreas como a biotecnologia e a engenharia genética possibilitam a produção de proteínas recombinantes, as quais podem ser utilizadas tanto na prevenção como no tratamento de doenças, sendo consideradas promissoras para o uso na indústria aquícola. Desta forma, fica evidente a necessidade de reunir toda informação referente ao estado da arte do uso de proteínas recombinantes na aquicultura. Assim, no primeiro capítulo dessa tese foi feita uma revisão do potencial terapêutico das proteínas imunorregulatórias na aquicultura, o que destaca que o uso de proteínas imunorregulatórias tem potencial para serem utilizadas na prevenção e tratamento de doenças de peixes. Dentre as proteínas que apresentam esse potencial, destaca-se as cistatinas. Desta forma, no segundo capítulo foi produzida uma Cistatina-B, oriunda da piranha vermelha Pygocentrus nattereri de forma heteróloga. As cistatinas são conhecidas por formar uma superfamília de inibidores de proteases de cisteína que apresentam um papel chave na degradação de proteínas e estão relacionadas a diferentes processos fisiológicos, como o desenvolvimento e imunidade. Para isso, juvenis de P. nattereri foram capturados no lago Janauacá, município de Manaquiri Amazonas, Brasil. Através da tecnologia do DNA recombinante a proteína Cistatina-B foi produzida e expressada na levedura Pichia pastoris. Foram avaliados a atividade biológica inibitória da protease de cisteína e ação bacteriostática nas bactérias Escherichia coli e Bacillus subtilis. A expressão e purificação da proteína imunorregulatória recombinante rCistatina-B revelou um fragmento de aproximadamente 11.8 kDa. A atividade inibitória atingiu 100% de inibição a uma concentração de 60 µg/µL resposta dependente-concentração do inibidor. Ação bacteriostática em E.coli e B. subtilis, numa concentração de 60 µg/µL de rCistatina-B, apresentou um halo de inibição de 9.66±0.55 mm e 10.50±2.54 mm, respectivamente, inferiores ao observado no controle positivo (espectinomicina) para as duas bactérias (23.93±1.42 e 21.39±1.83 mm) (P<0.05). Na concentração de 80 µg/µL a resposta foi similar, com valores de 8.83±0.72 mm para E.coli e 13.34±5.64 para B. subtilis, menores comparadas com o controle positivo (20±0.04 mm e 24.53±3.91) (P<0.05). Desta forma, o sistema de expressão eucarionte foi hábil em produzir a proteína recombinante rCistatina-B de Pygocentrus nattereri. A partir da confirmação do potencial biológico da Cistatina-B da piranha, análises in silico foram realizadas no genoma do peixe tambaqui Colossoma macropomum. Essas análises, que constituem o capítulo 3 dessa tese, permitiram identificar e analisar a Cistatina-B em todo o genoma do tambaqui. Foram identificados oito genes que codificam para a cistatina-B com base na sequência da piranha. Em geral, o tamanho das Cistatinas-B variaram de 96 a 101 aminoácidos, estão presentes majoritariamente no citoplasma, todas apresentaram motivos e domínios conservados nas cistatinas, além de apresentarem estrutura tridimensional similar a cistatina humana cristalografada. Como conclusão, o conhecimento e desenvolvimento das proteínas imunorregulatórias recombinantes e seu potencial terapêutico na aquicultura permite fornecer um novo bioproduto, o qual poderá ser usado como suplemento para estimulação do sistema imune em peixes.

Aquaculture is the fastest growing sector among animal production industries. Parallel to this important growth is the emergence of infectious diseases, which can cause significant losses in stocks and problems with animal welfare, being the biggest obstacle to advances in aquaculture production. To face this problem, it is necessary to associate good management techniques with the development of new technologies. Areas such as biotechnology and genetic engineering enable the production of recombinant proteins, which can be used both in the prevention and treatment of diseases, being considered promising for use in the aquaculture industry. Thus, it is evident the need to gather all information regarding the state of the art of the use of recombinant proteins in aquaculture. Thus, in the first chapter of this thesis, a review was made of the therapeutic potential of immunoregulatory proteins in aquaculture, which highlights that the use of immunoregulatory proteins has the potential to be used in the prevention and treatment of fish diseases. Among the proteins that have this potential, cystatins stand out. Thus, in the second chapter a Cystatin-B was produced, originating from the red piranha Pygocentrus nattereri in a heterologous way. Cystatins are known to form a superfamily of cysteine protease inhibitors that play a key role in protein degradation and are related to different physiological processes, such as development and immunity. For that, juveniles of P. nattereri were captured in the lake Janauacá, municipality of Manaquiri - Amazonas, Brazil. Through recombinant DNA technology, the protein Cystatin-B was produced and expressed in the yeast Pichia pastoris. The biological activity of cysteine protease and bacteriostatic action in Escherichia coli and Bacillus subtilis were evaluated. The expression and purification of the recombinant immunoregulatory protein rCystatin-B revealed a fragment of approximately 11.8 kDa. The inhibitory activity reached 100% inhibition at a concentration of 60 µg/µL response dependent on the concentration of the inhibitor. Bacteriostatic action in E.coli and B. subtilis, at a concentration of 60 µg /µL of rCystatin-B, showed an inhibition halo of 9.66 ± 0.55 mm and 10.50±2.54 mm, respectively, lower than that observed in the positive control (spectinomycin) for the two bacteria (23.93 ± 1.42 and 21.39 ± 1.83 mm) (P <0.05). At a concentration of 80 µg/µL the response was similar, with values of 8.83 ± 0.72 mm for E.coli and 13.34 ± 5.64 for B. subtilis, lower compared to the positive control (20 ± 0.04 mm and 24.53 ± 3.91) (P <0.05). Thus, the eukaryotic expression system was able to produce the recombinant protein rCystatin-B from Pygocentrus nattereri. From the confirmation of the biological potential of Cystatin-B of the piranha, in silico analyzes were carried out in the genome of the tambaqui fish Colossoma macropomum. These analyzes, which constitute chapter 3 of this thesis, made it possible to identify and analyze Cystatin-B throughout the tambaqui genome. Eight genes that code for Cystatin-B have been identified based on the piranha sequence. In general, the size of Cystatins-B varies from 96 to 101 amino acids, are present mainly in the cytoplasm, all of them presented motifs and domains conserved in cystatines, in addition to presenting a threedimensional structure similar to crystallized human cystatin. In conclusion, the knowledge and development of recombinant immunoregulatory proteins and their therapeutic potential in aquaculture allows us to provide a new bioproduct, which can be used as a supplement to stimulate the immune system in fish.

Performance of jundiá larvae, Rhamdia quelen, fed on probiotic supplemented diets / Desempenho de larvas de jundiá, Rhamdia quelen, alimentadas com dietas contendo probióticos

Since probiotics have proved to be a viable alternative to antibiotics as enhancers of animal growth, the performance, uniformity and mortality rates of the jundiá (Rhamdia quelen) larvae fed on diets with different probiotics were evaluated. Jundiá larvae, aged four days post hatching, were fed during 21 days with the following diets, in four replicates, namely, CO: control feed, without probiotics; PP: feed with Pichia pastoris; SB: feed with Saccharomyces boulardii; BT: feed with Bacillus cereus var. toyoi. Among the tested probiotic, Bacillus cereus var. toyoi had the best results due to the fact that the larvae were 25% heavier than CO at the end of the first week; the difference increased to 28% by the end of the trial. Further, BT also improved uniformity and Fultons condition factor. Larvae fed on Saccharomyces boulardii had the lowest body weight, whereas those fed on Pichia pastoris grew similarly to the control diet. Mortality rate was not affected by treatments. Bacillus cereus var. toyoi improves the performance and uniformity of the larvae, but does not affect mortality rate.

Devido aos probióticos terem se mostrado uma alternativa viável ao uso de antibióticos como promotores de crescimento para animais, foi avaliado o desempenho, uniformidade e taxa de mortalidade de larvas de jundiá alimentadas com dietas contendo diferentes probióticos. Larvas de jundiá, com quatro dias pós-eclosão, foram alimentadas durante 21 dias com as seguintes dietas em quatro repetições: CO: ração controle, sem adição de probiótico; PP: ração com Pichia pastoris; SB: ração com Saccharomyces boulardii e BT: ração com Bacillus cereus var. toyoi. Dentre os probióticos testados, Bacillus cereus var. toyoi apresentou os melhores resultados, pois ao final da primeira semana as larvas desse tratamento estavam 25% mais pesadas que as do controle e ao final do experimento a diferença aumentou para 28%. Além disso, BT também melhorou a uniformidade e o fator de condição. As larvas alimentadas com Saccharomyces boulardii apresentaram menor peso dentre todos os tratamentos. Pichia pastoris proporcionou às larvas um crescimento semelhante ao tratamento controle. Portanto, conclui-se que Bacillus cereus var. toyoi melhora o desempenho e a uniformidade de larvas de jundiá, porém não afeta a taxa de mortalidade

Performance of jundiá larvae, Rhamdia quelen, fed on probiotic supplemented diets / Desempenho de larvas de jundiá, Rhamdia quelen, alimentadas com dietas contendo probióticos

Since probiotics have proved to be a viable alternative to antibiotics as enhancers of animal growth, the performance, uniformity and mortality rates of the jundiá (Rhamdia quelen) larvae fed on diets with different probiotics were evaluated. Jundiá larvae, aged four days post hatching, were fed during 21 days with the following diets, in four replicates, namely, CO: control feed, without probiotics; PP: feed with Pichia pastoris; SB: feed with Saccharomyces boulardii; BT: feed with Bacillus cereus var. toyoi. Among the tested probiotic, Bacillus cereus var. toyoi had the best results due to the fact that the larvae were 25% heavier than CO at the end of the first week; the difference increased to 28% by the end of the trial. Further, BT also improved uniformity and Fultons condition factor. Larvae fed on Saccharomyces boulardii had the lowest body weight, whereas those fed on Pichia pastoris grew similarly to the control diet. Mortality rate was not affected by treatments. Bacillus cereus var. toyoi improves the performance and uniformity of the larvae, but does not affect mortality rate.(AU)

Devido aos probióticos terem se mostrado uma alternativa viável ao uso de antibióticos como promotores de crescimento para animais, foi avaliado o desempenho, uniformidade e taxa de mortalidade de larvas de jundiá alimentadas com dietas contendo diferentes probióticos. Larvas de jundiá, com quatro dias pós-eclosão, foram alimentadas durante 21 dias com as seguintes dietas em quatro repetições: CO: ração controle, sem adição de probiótico; PP: ração com Pichia pastoris; SB: ração com Saccharomyces boulardii e BT: ração com Bacillus cereus var. toyoi. Dentre os probióticos testados, Bacillus cereus var. toyoi apresentou os melhores resultados, pois ao final da primeira semana as larvas desse tratamento estavam 25% mais pesadas que as do controle e ao final do experimento a diferença aumentou para 28%. Além disso, BT também melhorou a uniformidade e o fator de condição. As larvas alimentadas com Saccharomyces boulardii apresentaram menor peso dentre todos os tratamentos. Pichia pastoris proporcionou às larvas um crescimento semelhante ao tratamento controle. Portanto, conclui-se que Bacillus cereus var. toyoi melhora o desempenho e a uniformidade de larvas de jundiá, porém não afeta a taxa de mortalidade(AU)

Use of ELISA based on NcSRS2 of Neospora caninum expressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs / Utilização de um ELISA baseado em NcSRS2 de Neospora caninum expressa em Pichia pastoris para diagnóstico de neosporose em ovinos e cães

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.(AU)

A neosporose é uma doença causada pelo protozoário Neospora caninum que leva a perdas econômicas importantes em muitos países. No presente estudo, é descrita a utilização da proteína recombinante NcSRS2 de N. caninum expressa em Pichia pastoris em um ensaio imunoenzimático indireto (ELISA) para o diagnóstico de infecção por Neospora em ovelhas e cães. Observou-se, que utilizando-se um ELISA, o teste produziu uma especificidade de 94,5% e uma sensibilidade de 100% para ovinos, e uma especificidade de 93,3% e sensibilidade de 100% para cães. Uma maior sensibilidade foi observada em relação à IFI que foi confirmada por Western blot. Os resultados deste estudo sugerem que a proteína recombinante expressa em P. pastoris é bom antígeno para ser utilizado no diagnóstico imunológico para detectar N. caninum em duas espécies importantes expostas a esta parasitose.(AU)

HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation

Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol -10 °C, 4X = 3% methanol -30 °C, and 5X = 1% methanol -10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris.

Expressão heteróloga da EMA-2 (equi merozoite antigen) de Theileria equi em Pichia pastoris com potencial utilização em imunobiológicos / Heterologous expression of EMA-2 (equi merozoite antigen) of Theileria equi in Pichia pastoris with potential use in immunobiologics

A piroplasmose equina causada por Theileria equi acomete os equinos de forma endêmica no Brasil e em diversos outros países tropicais e subtropicais. Considerada uma das mais importantes doenças de equinos, causa danos à saúde animal e perdas econômicas. A proteína equi merozoite antigen (EMA-2) é uma das principais proteínas de superfície, expressa nos diversos estágios do ciclo do parasita, estimula resposta imune em animais infectados, tornando-se um possível candidato para utilização em diagnóstico. O gene EMA-2 foi clonado e expresso na levedura Pichia pastoris. A proteína EMA-2 recombinante (rEMA-2) foi caracterizada antigenicamente por Western Blot e por ELISA indireto, utilizando-se soro de equino positivo para theileriose. O resultado do ELISA demonstrou uma especificidade de 90,9% e sensibilidade de 83,3%, quando comparado ao padrão, sendo superior à imunofluorescência (80,6% de especificidade e 75,0% de sensibilidade), o que sugere que a rEMA-2 expressa em P. pastoris é um promissor antígeno para ser utilizado como ferramenta no imunodiagnóstico de theileriose equina.(AU)

Theileria equi, the causative agent of equine piroplasmosis, is endemic in Brazil and many other tropical and subtropical countries. It is considered one of the most important diseases of horses causing animal health problems and significant economic loss. The Protein equi merozoite antigen-2 (EMA-2) is a major surface protein that is expressed in different parasite cycle stages and induces immune response in infected animals, being a possible candidate to be used in diagnose. EMA-2 gene was cloned and expressed in the yeast Pichia pastoris, and the recombinant protein EMA-2 (rEMA-2) was characterized by Western Blot and indirect ELISA using equine positive sera. The ELISA results demonstrated a specificity of 90.9% and a sensitivity of 83.3% compared to the standard ELISA and being superior to immunofluorescence (80.6% of specificity and 75.0% of sensitivity) suggesting that the rEMA-2 expressed in P. pastoris is a promising antigen to be used as a tool in immunodiagnostic of theileriasis.(AU)

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici

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.(AU)