RESUMEN
Escherichia coli strain HB101 harboring an expression plasmid bearing calf prochymosin gene under the control of the tac promoter was grown in the presence of IPTG with or without novobiocin at 28 and 40 degrees C, respectively. The differential rates of synthesis of prochymosin inclusions, and, for comparison, of beta-lactamase and beta-galactosidase, as well as plasmid copy number, were determined during the first hours of steady state growth. At 28 degrees C the induced expression of prochymosin gene was almost blocked. Addition of novobiocin did not alleviate this effect. In fact, it strengthened it, and we conclude that both these additive inhibitory effects are a consequence of the decrease in negative superhelical tension of plasmid DNA to an insufficient level. At 40 degrees C the differential rate of prochymosin synthesis was markedly enhanced. Since the copy number of the expression plasmid increased approximately to the same extent, we conclude that an increase in gene dose is the cause. The stimulation of cloned heterologous gene expression at 40 degrees C and inhibition at 28 degrees C may be conveniently used in biotechnological-scale cultivations of some recombinant bacteria.
Asunto(s)
Quimosina/biosíntesis , Precursores Enzimáticos/biosíntesis , Escherichia coli/genética , Calor , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Animales , Bovinos , Quimosina/genética , ADN Superhelicoidal/efectos de los fármacos , Precursores Enzimáticos/genética , Dosificación de Gen , Expresión Génica , Novobiocina/farmacología , Plásmidos/efectos de los fármacos , Inhibidores de Topoisomerasa IIAsunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Virus de la Mieloblastosis Aviar/enzimología , Virus del Sarcoma Aviar/enzimología , Endopeptidasas/metabolismo , Proteínas de Fusión gag-pol/metabolismo , Genes gag , Genes pol , Secuencia de Aminoácidos , Ácido Aspártico Endopeptidasas/biosíntesis , Ácido Aspártico Endopeptidasas/aislamiento & purificación , Virus de la Mieloblastosis Aviar/genética , Virus del Sarcoma Aviar/genética , Clonación Molecular/métodos , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/biosíntesis , Endopeptidasas/aislamiento & purificación , Escherichia coli , Proteínas de Fusión gag-pol/biosíntesis , Proteínas de Fusión gag-pol/aislamiento & purificación , Cinética , Datos de Secuencia Molecular , Regiones Terminadoras GenéticasRESUMEN
The high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies was achieved by optimization of solubilization and renaturation conditions. The solubilization was carried out in 8 M urea at various pHs, at various temperatures, and for various periods of time. The following values were found optimal: 1 h at 31 degrees C, pH 10.4. For successful correct refolding of solubilized prochymosin molecules it was found to be necessary to dilute the solution into an alkaline buffer (pH 10.7) in such a way that the final concentration of urea did not exceed 0.32 M and that of protein 0.275 mg/ml. Our optimized procedure gives about eight times higher yields of enzymatically active chymosin than the current published methods.