RESUMEN
A genetically structured mathematical model was developed and used to evaluate the influence of molecular parameters involved in the expression of a harmful recombinant protein (SPA::EcoRI). The system consists of the controlled expression of the endonuclease EcoRI cloned in the plasmid pMTC48. The control is exerted by the lambda CI repressor expressed from the plasmid pRK248cIts. The deleterious effect of the activity of the enzyme EcoRI on the host DNA is prevented by the action of the EcoRI methylase that is expressed constitutively from a third plasmid, pEcoR4. The model includes molecular mechanisms involved in the regulation of the expression of these genes and is used to determine cultural conditions that maximize the production of the recombinant protein.
Asunto(s)
Proteínas de Unión al ADN , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Modelos Genéticos , Plásmidos/genética , Proteína Estafilocócica A/biosíntesis , Simulación por Computador , Daño del ADN , Desoxirribonucleasa EcoRI/biosíntesis , Desoxirribonucleasa EcoRI/genética , Escherichia coli/clasificación , Escherichia coli/metabolismo , Ingeniería Genética/métodos , Cinética , Modelos Biológicos , Modelos Químicos , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad de la Especie , Proteína Estafilocócica A/genética , Staphylococcus/genética , Transcripción Genética , Proteínas Virales , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The expression of exoprotein synthesis of Staphylococcus aureus Sae mutant RC121 and its parental strain was studied under in vivo growth conditions. Cultures of both strains were inoculated into dialysis sacs implanted in sheep peritoneum. Results indicated that similar to in vitro grown mutant cells, Sae mutant RC121 shows diminished synthesis of alpha- and beta-hemolysin, coagulase, DNase and protein A. However, in vitro and in vivo grown mutant cultures showed different exoprotein profiles in SDS-PAGE; some bands from in vivo mutant cultures were diminished or missing and others appeared as more concentrated, when compared with the pattern of the in vivo grown parental strain, while all the exoprotein bands from the in vitro cultures of the mutant were diminished or missing as compared to the in vitro grown parental strain. The virulence of the Sae mutant, assayed by intraperitoneal injection in mice, was lower than that of the parental strain after both in vivo and in vitro growth conditions.