RESUMEN
Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.
Asunto(s)
Genes Bacterianos , Fosfolipasas A2/biosíntesis , Proteínas Recombinantes/biosíntesis , Streptomyces/enzimología , Sulfato de Amonio/metabolismo , Fusión Artificial Génica , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Secuencia de Bases , Precipitación Química , Cromatografía por Intercambio Iónico/métodos , Medios de Cultivo/metabolismo , Activación Enzimática , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Datos de Secuencia Molecular , Fosfolipasas A2/genética , Fosfolipasas A2/aislamiento & purificación , Plásmidos/genética , Plásmidos/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Streptomyces/genéticaRESUMEN
OBJECTIVES: The uptake of endogenous sterol from serum may allow Candida glabrata to survive azole treatment. This study aims to determine the contribution of a sterol transporter that alters fluconazole sensitivity in the presence of serum. METHODS: Bioinformatic analysis predicted CgAUS1 as the C. glabrata orthologue of the Saccharomyces cerevisiae transporters AUS1 and PDR11. To investigate whether the CgAUS1 gene has sterol transporter activity, we investigated the effects of an AUS1 deletion on the growth of a tetracycline-regulatable ERG9 strain (tet-ERG9aus1), wherein ERG9 expression is turned off giving rise to a sterol requirement. Tetracycline-dependent repression of CgAUS1 in the tet-AUS1 strain was used to determine the fluconazole susceptibility of CgAUS1 in the presence and absence of serum. RESULTS: The tetracycline-treated tet-ERG9aus1 strain failed to grow in the presence of serum, whereas the parental tet-ERG9AUS1 strain grew by incorporating sterol from exogenously supplied serum. Serum cholesterol protected cells against the antifungal effects of fluconazole and this protection was lost by repressing CgAUS1 gene expression. Furthermore, such protection was also observed during itraconazole treatment, but not observed in cells treated with non-azole antifungals. CONCLUSIONS: CgAUS1 appears to function as a sterol transporter that may contribute to lower azole susceptibility in the presence of serum and to protect C. glabrata against azole toxicity in vivo.