RESUMEN
A series of genes of proenterotoxin B from Staphylococcus aureus containing signal peptide mutant forms was constructed in order to study the functional roles of the introduced mutations. It was shown that a continuous mutation in the n-region of the signal peptide does not affect the secretion efficiency of proenterotoxin B, in contrast to the analogous mutation in the h-region. Point mutations of the proprotein signal peptide, including the N-terminal amino-acid residue of the mature protein, were obtained. It was shown that the introduced structural. changes cause a decrease in secretion efficiency and a redistribution of the protein in various compartments of Escherichia coli cells.
Asunto(s)
Enterotoxinas/química , Escherichia coli/genética , Precursores de Proteínas/química , Staphylococcus aureus/química , Secuencia de Aminoácidos , Membrana Celular/química , Membrana Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Enterotoxinas/genética , Enterotoxinas/metabolismo , Escherichia coli/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Periplasma/química , Periplasma/metabolismo , Mutación Puntual , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
A comparison of the primary structures of the protein translocation Sec-system proteins in the Shewanella oneidensis MR-1 and Escherichia coli bacteria was carried out. The process of translocation of recombinant pro-enteroxins (SEB and SEH) from Staphylococcus aureus and pro-streptavidin (SAV) from Streptomyces avidinii in the S. oneidensis MR-1 and E. coli cell periplasm was studied. It was demonstrated that these marker proteins are transferred into the periplasmic space of the S. oneidensis MR-1 transformant strain cells. The identity of N-terminal amino acid sequences of mature recombinant SEB, SEH, and SAV proteins (generated during post-translation proteolysis of leader peptide by the Sec-system both in E. coli and S. oneidensis MR-1) was established.
Asunto(s)
Sistemas de Secreción Bacterianos , Expresión Génica , Shewanella , Enterotoxinas/biosíntesis , Enterotoxinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Transporte de Proteínas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Shewanella/genética , Shewanella/metabolismoRESUMEN
Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacterium Shewanella oneidensis MR-1 (SoUP) was determined at 1.68â Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.
Asunto(s)
Proteínas Bacterianas/química , Shewanella/enzimología , Uridina Fosforilasa/química , Dominio Catalítico , Cristalografía por Rayos X , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Especificidad por Sustrato , Uridina/químicaRESUMEN
The streptavidin gene from Streptomyces avidinii was cloned, an expression plasmid constructed, and a highly effective strain producer of streptavidin created. It was shown that the leader peptide of streptavidin ensures the effective secretion of this protein into the periplasmic space of Escherichia coli cells. The degradation site of the leader peptide was detected. Upon treatment with the total fraction of proteases secreted by S. avidinii into the culture medium, "core" streptavidin was obtained, which retained the biotin-binding function.
Asunto(s)
Escherichia coli/metabolismo , Estreptavidina/genética , Streptomyces/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Genes Bacterianos , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Estreptavidina/metabolismoRESUMEN
A gene of human tumor-associated antigen VNTR(MUC1) bound to streptavidin, an expression plasmid, and a highly effective hybrid protein-producing strain were constructed. It was shown that the streptavidin leader peptide ensures an effective secretion of the hybrid protein into the periplasmic space of Escherichia coli cells. The hybrid protein was isolated in a homogeneous state and its immunogenic properties were studied.