Improvement of Endo-β-N-acetylglucosaminidase H production using silkworm-baculovirus protein expression system
Improvement of Endo-β-N-acetylglucosaminidase H production using silkworm-baculovirus protein expression system
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DOI：
10.1016/j.aspen.2015.01.006
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发表时间：
2015-06-01
影响因子：
1.5
通讯作者：
Lee, Jae Man
中科院分区：
文献类型：
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作者：
Masuda, Atsushi;Xu, Jian;Lee, Jae Man
内切-β-N-乙酰氨基葡萄糖苷酶H（Endo H）催化N-连接聚糖的壳二糖核心的GlcNAc残基之间的切割，留下一个GlcNAc残基连接到天冬酰胺上。内切H切割糖蛋白上的高甘露糖和杂合但不复杂的N-连接寡糖。由于其独特的特异性，Endo H被广泛用于糖蛋白的结构和功能分析。在我们以前的研究中，使用家蚕-杆状病毒表达系统生产了作为分泌蛋白的重组Endo H，但与大肠杆菌相比，产量较低（30 μ g Endo H/10 ml幼虫血淋巴）。本研究从表达不含外源信号肽的Endo H的重组杆状病毒感染的家蚕脂肪体中纯化了具有细胞内活性的重组Endo H。值得注意的是，产量（9.3毫克，从20蚕幼虫）是约310倍高，分泌到幼虫血淋巴中，如以前所报道的。此外，我们筛选了九州大学保存的家蚕品系，并鉴定了n17作为Endo H的高水平表达品系。(C)2015韩国应用昆虫学会、台湾昆虫学会、马来西亚植物保护学会。Elsevier B. V.出版，保留所有权利。
Endo-beta-N-acetylglucosaminidase H (Endo H) catalyzes cleavage between the GlcNAc residues of the chitobiose core of N-linked glycans, leaving one GlcNAc residues attached to asparagine. Endo H cleaves high mannose and hybrid, but not complex, N-linked oligosaccharides on glycoproteins. Because of its unique specificity, Endo H is widely used for the structural and functional analyses of glycoproteins. In our previous study, the recombinant Endo H was produced as a secreted protein using silkworm-baculovirus expression system, but the yield was low (30 mu g Endo H/10 ml larval hemolymph) compared to that of Escherichia coli. In this study, we purified active recombinant Endo H as an intracellular protein from fat body of silkworm infected with the recombinant baculovirus expressing Endo H without the exogenous signal peptide. Remarkably, the yield (9.3 mg from 20 silkworm larvae) was about 310-fold higher than that secreted into larval hemolymph as reported previously. In addition, we screened the silkworm strains maintained in Kyushu University and identified n17 as a high-level expression strain for Endo H. (C) 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.