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Biotechnol Lett. 2017 Sep;39(9):1299-1308. doi: 10.1007/s10529-017-2361-y. Epub 2017 May 25.

Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression.

Author information

1
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-Ku, Shizuoka, 422-8529, Japan.
2
Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-Ku, Shizuoka, 422-8529, Japan.
3
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-Ku, Nagoya, 467-8603, Japan.
4
Medical & Biological Laboratories Co., Ltd., 4-5-3 Sakae, Naka-Ku, Nagoya, 460-0008, Japan.
5
Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama Myodaiji, Okazaki, 444-8787, Japan.
6
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-Ku, Shizuoka, 422-8529, Japan. park.enoch@shizuoka.ac.jp.
7
Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-Ku, Shizuoka, 422-8529, Japan. park.enoch@shizuoka.ac.jp.

Abstract

OBJECTIVE:

To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae.

RESULTS:

Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man3GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins.

CONCLUSIONS:

Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

KEYWORDS:

BmNPV; IgG; MALDI-TOF-MS; N-Glycan; RNAi; Silkworm

PMID:
28547344
DOI:
10.1007/s10529-017-2361-y
[Indexed for MEDLINE]

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