Expression, purification and characterization of N-glycanase from Schizosaccharomyces pombe in Escherichia coli.
- Author:
Fengxue XIN
1
;
Peng WANG
;
Shenghua ZHONG
;
Qingsheng QI
Author Information
1. Science Department, Jiangxi Agriculture University, Nanchang 330045, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Glycosylation;
Hydrogen-Ion Concentration;
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase;
biosynthesis;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification;
Schizosaccharomyces;
enzymology;
genetics;
Temperature
- From:
Chinese Journal of Biotechnology
2008;24(4):592-597
- CountryChina
- Language:Chinese
-
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.
