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Expression, Purification, and Characterization of Endo-β-N-Acetylglucosaminidase H Using Baculovirus-Mediated Silkworm Protein Expression System

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Abstract

Endo-β-N-acetylglucosaminidase (Endo H) from Streptomyces plicatus hydrolyzes the core di-GlcNAc units of asparagine-linked oligosaccharides and is regarded as an important tool for glycobiology research. In the present study, we established a large-scale system to produce secreted Endo H using a silkworm-baculovirus expression system (silkworm-BES). The recombinant Endo H purified from silkworm hemolymph had activity comparable to that from recombinant Escherichia coli. As well as its well-characterized substrate RNase B, the Endo H from silkworm-BES was able to deglycosylate the high-mannose glycoproteins from silkworm hemolymph. Interestingly, the secretion amount of recombinant Endo H was significantly varied among the different silkworm strains, which could provide valuable information for larger-scale protein productions from silkworm-BES.

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Acknowledgments

The NIAS-Bm-oyanagi2 cell line for propagation of recombinant BmNPVs was kindly provided by Dr. Imanishi (National Institute of Agrobiological Sciences, Japan). We also thank Dr. Chisa Aoki (Kyushu University Graduate School) for providing the Bme21 cell line for the expression of recombinant protein. The MALDI TOF MS was kindly supported and analyzed by Center for Advanced Instrumental and Educational Supports (Faculty of Agriculture, Kyushu University).

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Authors and Affiliations

  1. Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan

    Takumi Mitsudome, Jian Xu, Yudai Nagata, Atsushi Masuda, Daisuke Morokuma, Zhiqing Li, Hiroaki Mon, Jae Man Lee & Takahiro Kusakabe

  2. Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan

    Kazuhiro Iiyama

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  1. Takumi Mitsudome
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  2. Jian Xu
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  3. Yudai Nagata
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  4. Atsushi Masuda
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  5. Kazuhiro Iiyama
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  6. Daisuke Morokuma
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  7. Zhiqing Li
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  8. Hiroaki Mon
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  9. Jae Man Lee
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  10. Takahiro Kusakabe
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Correspondence to Takahiro Kusakabe.

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Supplementary Fig. 1

Construction of the recombinant donor plasmid for generating baculoviruses using the Gateway LR reaction. After the Gateway LR reaction between the pENTRL2130K-EndoH-TEVH8STREP and pDEST8 vector, Endo H ORF was under the control of polyhedrin promoter (Polh) in the recombinant donor plasmid pDESTPolh30K-EndoH-TEVH8STREP and followed by an SV40 polyadenylation signal (SV40). L1, L2, R1, R2, B1, and B2, represent recombination sites for Gateway cloning. Cm chloramphenicol resistance gene, ccdB ccdB gene, 30K6G signal peptide from B. mori, Endo H Endo-β-N-acetylglucosaminidase, His × 8 8-histidine-tag, STREP Strep-tag (JPEG 479 kb)

Supplementary Fig. 2

Struture of N-linked oligosaccharides of RNase B. The filled squares and opened circles represent GlcNAc and mannose, respectively (JPEG 916 kb)

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Mitsudome, T., Xu, J., Nagata, Y. et al. Expression, Purification, and Characterization of Endo-β-N-Acetylglucosaminidase H Using Baculovirus-Mediated Silkworm Protein Expression System. Appl Biochem Biotechnol 172, 3978–3988 (2014). https://doi.org/10.1007/s12010-014-0814-5

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  • DOI: https://doi.org/10.1007/s12010-014-0814-5

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