RESUMEN
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.
Asunto(s)
Línea Celular , Medio de Cultivo Libre de Suero/farmacología , Drosophila melanogaster , Proteínas Recombinantes/metabolismo , Proteínas Virales de Fusión/aislamiento & purificación , Proteínas Virales de Fusión/metabolismo , Aminoácidos/metabolismo , Animales , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular/metabolismo , Línea Celular/fisiología , Proliferación Celular/efectos de los fármacos , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Virus de la Rabia/genética , Virus de la Rabia/metabolismo , Proteínas Recombinantes/genética , Factores de TiempoRESUMEN
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.
Asunto(s)
Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Virus Sincitial Respiratorio Humano/química , Proteínas Virales de Fusión/aislamiento & purificación , Proteínas Virales de Fusión/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología Estructural de Proteína , Proteínas Virales de Fusión/químicaRESUMEN
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.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Aphthovirus/genética , Escherichia coli/genética , Proteínas Virales de Fusión/aislamiento & purificación , Animales , Aphthovirus/inmunología , Western Blotting , Cápside/genética , Cápside/inmunología , Cápside/aislamiento & purificación , Proteínas de la Cápside , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Ratones , Pruebas de Neutralización , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunologíaRESUMEN
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