RESUMEN
Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application.
Asunto(s)
ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/aislamiento & purificación , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Clonación Molecular/métodos , Escherichia coli/genética , Exotoxinas/genética , Exotoxinas/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Factores de Virulencia/genética , Factores de Virulencia/aislamiento & purificación , ADP Ribosa Transferasas/metabolismo , Secuencia de Aminoácidos , Toxinas Bacterianas/metabolismo , Línea Celular , Supervivencia Celular , Codón/genética , Exotoxinas/metabolismo , Genes Bacterianos , Datos de Secuencia Molecular , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Factores de Virulencia/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMEN
We have purified an arginine-specific mono(ADP-ribosyl)transferase from chicken erythrocytes. The purified transferase was free from poly (ADP-ribose) polymerase activity. The molecular weight of the purified enzyme was estimated to be 27.5 kDa by gel filtration through Sephadex G-75 in a non-denaturing solvent. Activity gel experiments indicate that the active enzyme has an apparent molecular weight in SDS gels of about 28 kDa. The optimum pH of the reaction is about 8.0. The K(m) value for NAD+ of the purified enzyme is about 130 microM. Small molecular weight inhibitors of poly (ADP-ribose) polymerase have no significant effect on the mono ADP-ribosyl transferase enzyme activity. A number of inhibitors of the arginine-specific mono(ADP-ribosyl)transferase activity have been identified. Among the more effective inhibitors are 1,4 naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 4-amino-1-naphthol and 1,2-naphthoquinone. We have also cloned a mono(ADP-ribosyl)transferase from chicken erythroblasts. This gene has been expressed in E. coli and ADP-ribosylation activity has been demonstrated using histones as substrate. The activity is shown to be arginine-specific by the use of poly-L-arginine as substrate. Use of a specific inhibitor has shown that this enzyme is indeed a mono(ADP-ribosyl)transferase and not a NAD glycohydrolase activity. The sequence of this gene is very similar to several other mono(ADP-ribosyl)transferase genes. There are thus at least three different chicken mono(ADP-ribosyl)transferase genes in the blood system alone; this suggests that there is a quite large family of mono(ADP-ribosyl)transferase genes in animals. We have also isolated the promoter region of this chicken gene and are able to identify several standard motifs in this promoter.