| RRC ID |
34803
|
| 著者 |
Masuda A, Xu J, Mitsudome T, Nagata Y, Morokuma D, Mon H, Banno Y, Kusakabe T, Lee JM.
|
| タイトル |
Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System.
|
| ジャーナル |
Mol Biotechnol
|
| Abstract |
The peptide-N (4)-(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.
|
| 巻・号 |
57(8)
|
| ページ |
735-45
|
| 公開日 |
2015-8-1
|
| DOI |
10.1007/s12033-015-9866-1
|
| PMID |
25832992
|
| MeSH |
Animals
Baculoviridae*
Bombyx*
Cell Line
Gene Expression*
Insect Proteins / biosynthesis*
Insect Proteins / genetics
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / biosynthesis*
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / genetics
Recombinant Proteins / biosynthesis
Recombinant Proteins / genetics
|
| IF |
2.022
|
| 引用数 |
11
|
|
WOS 分野
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| カイコ |
a49
a60
c51
c60
d17
d36
f38
g32
l80
l90
m90
n17
p50
p53
p62
t32
t70 |
