Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris
| Autor: | Bo Chen, Yi-Feng Pan, Da Lei, Liang Xiong, Qinghua He, Yanping Li, Yang Xu |
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| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular Glycosylation medicine.medical_treatment Aspergillus oryzae Molecular Sequence Data Bioengineering Applied Microbiology and Biotechnology Pichia Pichia pastoris law.invention Fungal Proteins chemistry.chemical_compound N-linked glycosylation law Tandem Mass Spectrometry medicine Amino Acid Sequence Protease biology Chemistry Metalloendopeptidases General Medicine biology.organism_classification Trypsin Molecular biology Recombinant Proteins carbohydrates (lipids) Biochemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Recombinant DNA Chromatography column Biotechnology medicine.drug Chromatography Liquid |
| Zdroj: | Biotechnology letters. 35(12) |
| ISSN: | 1573-6776 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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