RESUMO
The recombinant G glycoprotein from the surface of the rabies virus (RVGP) is a promising candidate as a rabies vaccine component and also for diagnostic purposes. In this study, RVGP production by transfected Drosophila melanogaster S2 cells cultivated in a serum-free medium (supplemented IPL-41 medium) was carried out. The effects of pH and pO(2) were evaluated in batch culture in parallel spinner flasks. The use of a pH equal to 6.3 and a pO(2) of 40% air saturation resulted in the highest RVGP content. These conditions were also used in fed-batch mode, yielding a RVGP content level of 98g/10(7) cells. The main nutrients consumed were glucose, glutamine, asparagine, serine and proline and the major metabolites produced were alanine and ammonia, according to the metabolism studies performed. Since RVGP is a transmembrane protein, two different methods for protein recovery were assessed and compared. Detergent-based cell disruption showed to be more effective than mechanical disruption with glass beads for glycoprotein recovery.
Assuntos
Linhagem Celular , Meios de Cultura Livres de Soro/farmacologia , Drosophila melanogaster , Proteínas Recombinantes/metabolismo , Proteínas Virais de Fusão/isolamento & purificação , Proteínas Virais de Fusão/metabolismo , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular/metabolismo , Linhagem Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Proteínas Recombinantes/genética , Fatores de TempoRESUMO
The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia coli BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. coli and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies.
Assuntos
Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Vírus Sincicial Respiratório Humano/química , Proteínas Virais de Fusão/isolamento & purificação , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , Homologia Estrutural de Proteína , Proteínas Virais de Fusão/químicaRESUMO
1. cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed. 2. The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed. 3. When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3. 4. Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro.
Assuntos
Anticorpos Antivirais/biossíntese , Aphthovirus/genética , Escherichia coli/genética , Proteínas Virais de Fusão/isolamento & purificação , Animais , Aphthovirus/imunologia , Western Blotting , Capsídeo/genética , Capsídeo/imunologia , Capsídeo/isolamento & purificação , Proteínas do Capsídeo , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Camundongos , Testes de Neutralização , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
1. cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed. 2. The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed. 3. When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3. 4. Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro