RESUMO
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.
Assuntos
Proteínas de Ligação a DNA , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Modelos Genéticos , Plasmídeos/genética , Proteína Estafilocócica A/biossíntese , Simulação por Computador , Dano ao DNA , Desoxirribonuclease EcoRI/biossíntese , Desoxirribonuclease EcoRI/genética , Escherichia coli/classificação , Escherichia coli/metabolismo , Engenharia Genética/métodos , Cinética , Modelos Biológicos , Modelos Químicos , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Proteína Estafilocócica A/genética , Staphylococcus/genética , Transcrição Gênica , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
Analysis of the chromosomic beta-galactosidase activity in strains of Escherichia coli with and without plasmids indicated that plasmid maintenance enhances gene expression. Cyclic AMP (cAMP) determinations confirmed that the gene enhancement observed in strains carrying plasmids was due to a small increase in the intracellular concentration of cAMP. Also, cells carrying plasmids displayed higher specific glucose uptake rates than did cells without plasmids. The increases in the expression of beta-galactosidase and the glucose uptake rate suggest a cAMP-mediated release of the glucose effect due to plasmid maintenance. Our results suggested that this effect is independent of the host and type and number of plasmids.