Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System

Atsushi Masuda, Jian Xu, Takumi Mitsudome, Yudai Nagata, Daisuke Morokuma, Hiroaki Mon, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The peptide-N4-(N-acetyl-β-d-glucosaminyl) asparagine amidase F (PNGase F) catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from glycoproteins. The PNGase F has broad substrate specificity and thus is extensively used for the structural and functional studies of the glycoproteins. In this study, we tried to produce active recombinant PNGase F as secreted and intracellular-expressed forms using baculovirus expression vector system (BEVS) through silkworm larvae or cultured cells. PNGase F itself contains potential N-linked glycosylation sites and we found that it was N-glycosylated when PNGase F secreted from silkworm cells. Intriguingly, the secreted recombinant PNGase F has the lower catalytic activity and self-digests its N-linked glycans and therefore this secreted form of this enzyme produced from BEVS is not appropriate for carbohydrate chain analysis. Instead, we successfully mass-produced (2.1 mg/20 silkworm larvae) and purified active recombinant PNGase F as an intracellular protein without N-glycosylations. Besides, we confirmed by directed mutagenesis that several amino acid residues are crucial for the function of PNGase F. Our results provide an alternative method for the mass production of active enzymes involved in the study of glycoproteins.

Original languageEnglish
Pages (from-to)735-745
Number of pages11
JournalMolecular Biotechnology
Volume57
Issue number8
DOIs
Publication statusPublished - Aug 25 2015

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Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Glycoproteins
Bombyx
Baculoviridae
Peptides
Glycosylation
Oligosaccharides
Enzymes
Mutagenesis
Carbohydrates
Amino acids
Asparagine
Catalyst activity
amidase
Cells
Proteins
Larva
Substrates
Glycoside Hydrolases

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

Cite this

Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System. / Masuda, Atsushi; Xu, Jian; Mitsudome, Takumi; Nagata, Yudai; Morokuma, Daisuke; Mon, Hiroaki; Banno, Yutaka; Kusakabe, Takahiro; Lee, Jae Man.

In: Molecular Biotechnology, Vol. 57, No. 8, 25.08.2015, p. 735-745.

Research output: Contribution to journalArticle

Masuda, Atsushi ; Xu, Jian ; Mitsudome, Takumi ; Nagata, Yudai ; Morokuma, Daisuke ; Mon, Hiroaki ; Banno, Yutaka ; Kusakabe, Takahiro ; Lee, Jae Man. / Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System. In: Molecular Biotechnology. 2015 ; Vol. 57, No. 8. pp. 735-745.
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