A transgenic insect cell line engineered to produce CMP-sialic acid and sialylated glycoproteins
A transgenic insect cell line engineered to produce CMP-sialic acid and sialylated glycoproteins
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DOI：
10.1093/glycob/cwg051
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发表时间：
2003-06-01
期刊：
影响因子：
4.3
通讯作者：
Jarvis, DL
中科院分区：
文献类型：
--
作者：
Aumiller, JJ;Hollister, JR;Jarvis, DL
我们先前已经设计了转基因昆虫细胞系来表达哺乳动物糖基转移酶，并表明这些细胞可以唾液酸化N-糖蛋白，尽管它们几乎没有细胞内唾液酸并且没有可检测到的CMP-唾液酸。在随后的研究中，我们提出的证据表明，这些细胞系可以挽救唾液酸从头糖蛋白唾液酸化细胞外唾液酸辅助蛋白，如胎球蛋白，发现在胎牛血清。这一发现使我们创造了一种新的转基因昆虫细胞系，旨在合成自己的唾液酸和CMP-唾液酸。用编码唾液酸合成酶和CMP-唾液酸合成酶的另外两种哺乳动物基因转化编码五种哺乳动物糖基转移酶的SfSWT-1细胞。所得细胞系表达所有7种哺乳动物基因，产生CMP-唾液酸，并在无血清生长培养基中培养时，唾液酸化的重组糖蛋白补充N-乙酰甘露糖胺。因此，添加编码两种参与CMP-唾液酸生物合成的酶的哺乳动物基因产生了新的转基因昆虫细胞系SfSWT-3，其可以在不存在胎牛血清的情况下唾液酸化重组糖蛋白。这一新的细胞系将广泛用作杆状病毒介导的重组糖蛋白生产的改良宿主。
We have previously engineered transgenic insect cell lines to express mammalian glycosyltransferases and showed that these cells can sialylate N-glycoproteins, despite the fact that they have little intracellular sialic acid and no detectable CMP-sialic acid. In the accompanying study, we presented evidence that these cell lines can salvage sialic acids for de novo glycoprotein sialylation from extracellular sialogly-coproteins, such as fetuin, found in fetal bovine serum. This finding led us to create a new transgenic insect cell line designed to synthesize its own sialic acid and CMP-sialic acid. SfSWT-1 cells, which encode five mammalian glycosyltransferases, were transformed with two additional mammalian genes that encode sialic acid synthase and CMP-sialic acid synthetase. The resulting cell line expressed all seven mammalian genes, produced CMP-sialic acid, and sialylated a recombinant glycoprotein when cultured in a serum-free growth medium supplemented with N-acetylmannosamine. Thus the addition of mammalian genes encoding two enzymes involved in CMP-sialic acid biosynthesis yielded a new transgenic insect cell tine, SfSWT-3, that can sialylate recombinant glycoproteins in the absence of fetal bovine serum. This new cell line will be widely useful as an improved host for baculovirus-mediated recombinant glycoprotein production.