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首页> 外文期刊>Biotechnology Progress >Identification of Genes Encoding N-glycan Processing β-N-acetylgIucosaminidases in Trichoplusia ni and Bombyx mori: Implications for Glycoengineering of Baculovirus Expression Systems
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Identification of Genes Encoding N-glycan Processing β-N-acetylgIucosaminidases in Trichoplusia ni and Bombyx mori: Implications for Glycoengineering of Baculovirus Expression Systems

机译：毛滴虫和家蚕中编码N-聚糖加工β-N-乙酰基葡糖苷酶的基因的鉴定：对杆状病毒表达系统糖工程的意义

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摘要

Glycoproteins produced by non-engineered insects or insect cell lines characteristically bear truncated, paucimannose N-glycans in place of the complex N-glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N-gly-can processing β-N-acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N-glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing β-N-acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Eioinformatic analyses of putative processing β-N-acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing β-N-acetylglucosaminidases than degradative or chitinolytic β-N-acetylglucosaminidases. In addition, over-expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing β-N-acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus-insect cell expression systems for the production of glycosylated proteins.
机译：非工程昆虫或昆虫细胞系产生的糖蛋白通常带有截短的甘露糖N-聚糖，以代替哺乳动物细胞产生的复杂N-聚糖。造成这种差异的主要原因是昆虫中存在高度特异性的N-甘氨酸加工β-N-乙酰氨基葡糖苷酶，而哺乳动物系统中没有。因此，减少或消除这种酶可以增强糖工程昆虫或昆虫细胞系产生复杂N-聚糖的能力。在常规用于重组糖蛋白生产的三种昆虫中，仅从节食夜蛾（Spodoptera frugiperda）中分离到加工性β-N-乙酰氨基葡萄糖苷酶基因。因此，本研究的目的是从其他两个物种家蚕和毛癣菌中分离并鉴定编码这种重要加工酶的基因。从这两个物种分离的推定加工β-N-乙酰氨基葡萄糖苷酶基因的电子信息学分析表明，每个编码的产物的确比降解或几丁质β-N-乙酰氨基葡萄糖苷酶更类似于加工β-N-乙酰氨基葡萄糖苷酶。另外，这些基因中的每一个的过表达都诱导了具有加工的底物特异性特征的酶活性，而不是降解或几丁质酶。总之，这些结果表明，已经成功地从毛滴虫和家蚕中分离出了加工β-N-乙酰氨基葡糖苷酶基因。这些基因的鉴定具有促进杆状病毒-昆虫细胞表达系统的进一步糖工程化以产生糖基化蛋白质的潜力。

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