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 language | English |
|---|---|
| Pages (from-to) | 735-745 |
| Number of pages | 11 |
| Journal | Molecular Biotechnology |
| Volume | 57 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - Aug 25 2015 |
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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, Man.
In: Molecular Biotechnology, Vol. 57, No. 8, 25.08.2015, p. 735-745.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System
AU - Masuda, Atsushi
AU - Xu, Jian
AU - Mitsudome, Takumi
AU - Nagata, Yudai
AU - Morokuma, Daisuke
AU - Mon, Hiroaki
AU - Banno, Yutaka
AU - Kusakabe, Takahiro
AU - Lee, Man
PY - 2015/8/25
Y1 - 2015/8/25
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=84937978503&partnerID=8YFLogxK
U2 - 10.1007/s12033-015-9866-1
DO - 10.1007/s12033-015-9866-1
M3 - Article
VL - 57
SP - 735
EP - 745
JO - Molecular Biotechnology
JF - Molecular Biotechnology
SN - 1073-6085
IS - 8
ER -