Expression, purification and characterization of N-glycanase from Schizosaccharomyces pombe in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 592-597, 2008.
Artículo
en Chino
| WPRIM
| ID: wpr-342865
ABSTRACT
One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b(+)/Png1p was transformed into E. coli BL21(DE3). The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Png1p in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30 degrees C; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100 degrees C.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Schizosaccharomyces
/
Temperatura
/
Glicosilación
/
Proteínas Recombinantes
/
Clonación Molecular
/
Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa
/
Escherichia coli
/
Genética
/
Concentración de Iones de Hidrógeno
/
Metabolismo
Idioma:
Chino
Revista:
Chinese Journal of Biotechnology
Año:
2008
Tipo del documento:
Artículo
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