RESUMEN
In the previous study we cloned Pyrococcus woesei gene coding thermostable beta-galactosidase into pET30-LIC expression plasmid. The nucleotide sequence revealed that beta-galactosidase of P. woesei consists of 510 amino acids and has a molecular weight of 59, 056 kDa (GenBank Accession No. AF043283). It shows 99.9% nucleotide identity to the nucleotide sequence of beta-galactosidase from Pyrococcus furiosus. We also demonstrated that thermostable beta-galactosidase can be produced with high yield by Escherichia coli strain and can be easy separated by thermal precipitation of other bacterial proteins at 85 degrees C (S. D $$;abrowski, J. Maciunska, and J. Synowiecki, 1998, Mol. Biotechnol. 10, 217-222). In this study we presented a new expression system for producing P. woesei beta-galactosidase in Escherichia coli and one-step chromatography purification procedure for obtaining pure enzyme (His(6)-tagged beta-galactosidase). The recombinant beta-galactosidase contained a polyhistidine tag at the N-terminus (20 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. The enzyme was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni(2+)-TED-Sepharose columns. The enzyme was characterized and displayed high activity and thermostability. This bacterial expression system appears to be a good method for production of the thermostable beta-galactosidase.
Asunto(s)
Pyrococcus/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , beta-Galactosidasa/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía en Agarosa , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Calefacción , Histidina/química , Datos de Secuencia Molecular , Níquel , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sefarosa/análogos & derivados , beta-Galactosidasa/química , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The 3' part of the glucosamine-6-phosphate synthase gene from Histoplasma capsulatum was PCR amplified using degenerate primers designed from the known glucosamine-6-phosphate synthase gene sequences, cloned and sequenced. The computer analysis of the 676 bp sequence revealed the presence of two introns. The identities of the deduced amino acid sequence to the corresponding Saccharomyces cerevisiae and Candida albicans fragment are 65 and 63.8%, respectively.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Histoplasma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Histoplasma/enzimología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Alineación de SecuenciaRESUMEN
The proposed here PCR thermal profile improves the specificity and efficiency of PCR using highly degenerate primers, especially in the case of larger PCR products (around 2000 bp and more). The improvement is achieved by the use of a specific annealing temperature in the beginning cycles and the alternate lowering and raising of the annealing temperature in the subsequent cycles.