Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content.

Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures.

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Cost analysis based on bioreactor cultivation conditions: Production of a soluble recombinant protein using Escherichia coli BL21(DE3).

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32 °C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Cost analysis based on bioreactor cultivation conditions: production of a soluble recombinant protein using Escherichia coli BL21(DE3)

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32°C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

Oral lichen planus with cutaneous manifestations: case report with emphasis on dental diagnostic criteria / Líquen plano oral com manifestações cutâneas: relato de caso com ênfase nos critérios de diagnóstico odontológico

ABSTRACT Lichen planus is a chronic inflammatory disease involving the skin and mucosa, which often affects the oral cavity. The objective of this study is to report a case of oral lichen planus (OLP) with cutaneous manifestations and to discuss the clinical, histopathological aspects and the established treatment. A 61-years-old female white patient was referred for evaluation of white lesions in the oral mucosa. In the intraoral examination, multiple white lesions with striated appearance were observed in the jugal mucosa, tongue and border. The extraoral examination revealed scaly lesions on the arm, white spots on the legs, and nail dystrophy on feet. Based on biopsy of the oral lesions and the histopathological analysis, the diagnosis of OLP was confirmed. The patient underwent treatment with clobetasol propionate topical cream (0.5 mg), and was instructed to apply it to affected area, once or twice a day for four weeks. In the clinical follow-up after one month and 15 days, the improvement of the lesions could be analyzed. As OLP is a disease with an etiopathogenesis that is still poorly recognized, several factors may enable the development of this condition. Therefore, the dentist's clinical view is essential for the most effective treatment.

RESUMEN El liquen plano es una enfermedad inflamatoria crónica que involucra piel y mucosa, con frecuencia de la cavidad bucal. Los objetivos de este estudio son reportar un caso de liquen plano oral (LPO) con manifestaciones cutáneas y discutir sus aspectos clínicos e histopatológico, así como el tratamiento establecido. Reportamos el caso de una mujer de 61 años de edad, raza blanca, que fue remitida para evaluación de lesiones blancas en la mucosa bucal. En el examen intraoral se observaron lesiones blancas múltiples estriadas en mucosa yugal, lengua y cresta; en el examen extraoral, lesiones descamativas en el brazo, manchas blancas en las piernas y uñas distróficas en los pies. Basándose en la biopsia de las lesiones bucales y en el análisis histopatológico, el diagnóstico de LPO se confirmó. La paciente fue sometida a tratamiento con propionato de clobetasol crema (0,5 mg) y aconsejada a aplicar el medicamento sobre el área afectada, una o dos veces al día, por cuatro semanas. En el seguimiento clínico, al cabo de un mes y 15 días, fue posible analizar la mejoría de las lesiones. Por tratarse de una enfermedad de etiología todavía poco conocida, varios factores pueden favorecer su desarrollo. Así, el ojo clínico del cirujano dentista es imprescindible para un tratamiento más efectivo.

RESUMO O líquen plano é uma doença inflamatória crônica que envolve pele e mucosa, acometendo frequentemente a cavidade bucal. Os objetivos deste estudo são relatar um caso de líquen plano oral (LPO) com manifestações cutâneas e discutir os aspectos clínicos e histopatológicos, bem como o tratamento estabelecido. Relatamos o caso de uma mulher, 61 anos de idade, leucoderma, que foi encaminhada para avaliação de lesões brancas na mucosa bucal. Ao exame intraoral, foram observadas múltiplas lesões brancas com aspecto estriado em mucosa jugal, língua e rebordo; ao exame extraoral, lesões de aspecto descamativo no braço, manchas brancas nas pernas e unhas distróficas nos pés. Com base na biópsia das lesões bucais e na análise histopatológica, o diagnóstico de LPO foi confirmado. A paciente foi submetida ao tratamento com propionato de clobetasol em creme (0,5 mg) e orientada a fazer a aplicação na área afetada, uma a duas vezes ao dia, durante quatro semanas. No acompanhamento clínico após um mês e 15 dias, pôde-se analisar a melhora das lesões. Por se tratar de uma doença com etiopatogênese ainda pouco reconhecida, vários fatores podem possibilitar o desenvolvimento dessa condição. Dessa forma, é imprescindível o olhar clínico do cirurgião-dentista para o tratamento mais eficaz.

Salmonella typhimurium and Escherichia coli dissimilarity: Closely related bacteria with distinct metabolic profiles.

Live attenuated strains of Salmonella typhimurium have been extensively investigated as vaccines for a number of infectious diseases. However, there is still little information available concerning aspects of their metabolism. S. typhimurium and Escherichia coli show a high degree of similarity in terms of their genome contents and metabolic networks. However, this work presents experimental evidence showing that significant differences exist in their abilities to direct carbon fluxes to biomass and energy production. It is important to study the metabolism of Salmonella to elucidate the formation of acetate and other metabolites involved in optimizing the production of biomass, essential for the development of recombinant vaccines. The metabolism of Salmonella under aerobic conditions was assessed using continuous cultures performed at dilution rates ranging from 0.1 to 0.67 h(-1), with glucose as main substrate. Acetate assimilation and glucose metabolism under anaerobic conditions were also investigated using batch cultures. Chemostat cultivations showed deviation of carbon towards acetate formation, starting at dilution rates above 0.1 h(-1). This differed from previous findings for E. coli, where acetate accumulation was only detected at dilution rates exceeding 0.4 h(-1), and was due to the lower rate of acetate assimilation by S. typhimurium under aerobic conditions. Under anaerobic conditions, both microorganisms mainly produced ethanol, acetate, and formate. A genome-scale metabolic model, reconstructed for Salmonella based on an E. coli model, provided a poor description of the mixed fermentation pattern observed during Salmonella cultures, reinforcing the different patterns of carbon utilization exhibited by these closely related bacteria.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Clinical-pathological aspects of oral lymphoepithelial cyst: case report / Aspectos clinicopatológicos de cisto linfoepitelial oral: relato de caso

ABSTRACT Oral lymphoepithelial cyst (OLEC) is an uncommon lesion whose pathogenesis remains poorly understood. The purpose of this paper is to report a case of OLEC. Female patient, white, 62 years old, presented asymptomatic nodular swelling of soft consistency in the lateral border of the tongue. Under the clinical hypothesis of lymphoid tissue hyperplasia, an excisional biopsy was performed. Histopathological examination revealed a pathological epithelial-lined cavity and a cystic connective tissue capsule containing lymphoid tissue. The diagnosis of OLEC was established and the patient showed no signs of recurrence after surgical removal of the lesion.

RESUMEN El quiste linfoepitelial (QLE) oral es una lesión infrecuente, cuya patogénesis es aún poco conocida. El objetivo del presente estudio es reportar un caso de QLE oral. Mujer blanca de 62 años presentó un crecimiento nodular asintomático de consistencia blanda, en borde lateral de la lengua. Bajo la hipótesis clínica de hiperplasia del tejido linfoide, se realizó una biopsia excisional. El examen histopatológico reveló cavidad patológica revestida de epitelio y una cápsula quística de tejido conectivo, conteniendo tejido linfoide. Se estableció el diagnóstico de QLE oral. La paciente no ha presentado recidiva tras extirpación quirúrgica de la lesión.

RESUMO O cisto linfoepitelial oral (CLEO) é uma lesão incomum, cuja patogênese ainda é pouco elucidada. O objetivo deste estudo é relatar um caso de CLEO. Paciente do sexo feminino, leucoderma, 62 anos, apresentou aumento de volume nodular assintomático de consistência amolecida, em borda lateral da língua. Sob a hipótese clínica de hiperplasia de tecido linfoide, biópsia excisional foi realizada. O exame histopatológico revelou cavidade patológica revestida por epitélio e uma cápsula cística de tecido conjuntivo, contendo tecido linfoide. O diagnóstico de CLEO foi estabelecido. A paciente não apresentou sinais de recidiva após a remoção cirúrgica da lesão.

Cost analysis based on bioreactor cultivation conditions: production of a soluble recombinant protein using Escherichia coli BL21(DE3)

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32°C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content

Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 A degrees C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 +/- 2.5% of PspA4Pro with 97.8 +/- 0.36% purity and reduced endotoxin concentration by > 99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.