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Protein Expr Purif ; 81(2): 145-50, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22019762

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/genética
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