Abstract
Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff’s base staining and Endo H digestion. Moreover, the deglycosylated protein’s molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI–TOF–MS, and the hyperglycosylation extent was 21 %. The N-glycosylation site of rNPI was analyzed by nano LC–MS/MS after digesting by trypsin and Glu-C, and the unique potential site Asn41 of mature peptide was found to be glycosylated. Homology modeling of the 3D structure of rNPI indicated that the attached N-glycans hardly affected neutral protease’s activity due to the great distance away from the active site of the enzyme.
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This work has been supported by the National Natural Science Foundation of P. R. China (Project No. 31260389).
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Lei, D., Xu, Y., He, Q. et al. Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris . Biotechnol Lett 35, 2121–2127 (2013). https://doi.org/10.1007/s10529-013-1314-3
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DOI: https://doi.org/10.1007/s10529-013-1314-3