Biotechnology Letters

, Volume 35, Issue 12, pp 2121–2127 | Cite as

Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris

  • Da Lei
  • Yang Xu
  • Qinghua He
  • Yifeng Pan
  • Bo Chen
  • Liang Xiong
  • Yanping Li
Original Research Paper

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.

Keywords

Aspergillus oryzae N-glycosylation Nano LC–MS/MS Pichia pastoris Protease Recombinant neutral protease I 

References

  1. Arnold K, Bordoli L, Kopp J et al (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201PubMedCrossRefGoogle Scholar
  2. Carlsson SR (1993) Isolation and characterization of glycoproteins. In: Fukuda M, Kobata A (eds) Glycobiology, a practical approach. Oxford University Press Inc., New York, pp 1–26Google Scholar
  3. Cupp-Enyard C (2008) Sigma’s non-specific protease activity assay—casein as a substrate. J Vis Exp 19:899–900PubMedGoogle Scholar
  4. De Pourcq K, De Schutter K, Callewaert N et al (2010) Engineering of glycosylation in yeast and other fungi: current state and perspectives. Appl Microbiol Biotechnol 87:1617–1631PubMedCrossRefGoogle Scholar
  5. Dean N (1999) Asparagine-linked glycosylation in the yeast golgi. BBA-Gen Subj 1426:309–322CrossRefGoogle Scholar
  6. Gomis-Rüth FX, Botelho TO, Bode W (2012) A standard orientation for metallopeptidases. BBA-Proteins Proteomics 1824:157–163PubMedCrossRefGoogle Scholar
  7. Grinna LS, Tschopp JF (1989) Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast 5:107–115PubMedCrossRefGoogle Scholar
  8. Guo M, Hang H, Zhu T et al (2008) Effect of glycosylation on biochemical characterization of recombinant phytase expressed in Pichia pastoris. Enzym Microb Technol 42:340–345CrossRefGoogle Scholar
  9. Hsiao ES, Chen JC, Tsai HY et al (2009) Determination of N-glycosylation site and glycan structures of pectin methylesterase in jelly fig (Ficus awkeotsang) achenes. J Agric Food Chem 57:6757–6763PubMedCrossRefGoogle Scholar
  10. Ke Y, Huang WQ, Li JZ et al (2012) Enzymatic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris. J Agric Food Chem 60:12164–12169PubMedCrossRefGoogle Scholar
  11. Machida M, Asai K, Sano M et al (2005) Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161PubMedCrossRefGoogle Scholar
  12. Montesino R, Garcia R, Quintero O et al (1998) Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris. Protein Expr Purif 14:197–207PubMedCrossRefGoogle Scholar
  13. Tatsumi H, Murakami S, Tsuji R et al (1991) Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease. Mol Gen Genet 228:97–103PubMedCrossRefGoogle Scholar
  14. Wang LH, Li DQ, Fu Y et al (2007) pFind 2.0: a software package for peptide and protein identification via tandem mass spectrometry. Rapid Commun Mass Spectrom 21:2985–2991PubMedCrossRefGoogle Scholar
  15. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Da Lei
    • 1
  • Yang Xu
    • 1
    • 2
  • Qinghua He
    • 1
    • 2
  • Yifeng Pan
    • 1
  • Bo Chen
    • 1
  • Liang Xiong
    • 1
  • Yanping Li
    • 1
    • 2
  1. 1.State Key Laboratory of Food Science and TechnologyNanchang UniversityNanchangChina
  2. 2.Jiangxi-OAI Joint Research InstituteNanchang UniversityNanchangChina

Personalised recommendations