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

研究成果: Contribution to journalArticle査読

12 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)735-745
ページ数11
ジャーナルMolecular Biotechnology
57
8
DOI
出版ステータス出版済み - 8 25 2015

All Science Journal Classification (ASJC) codes

  • バイオテクノロジー
  • バイオエンジニアリング
  • 生化学
  • 応用微生物学とバイオテクノロジー
  • 分子生物学

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