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.
Article
in Chinese
| 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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Schizosaccharomyces
/
Temperature
/
Glycosylation
/
Recombinant Proteins
/
Cloning, Molecular
/
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
/
Escherichia coli
/
Genetics
/
Hydrogen-Ion Concentration
/
Metabolism
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2008
Type:
Article
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