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首页> 外文期刊>Biotechnology Progress >Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins
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Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins

机译：新型昆虫细胞系能够进行复杂的N-糖基化和重组蛋白唾液酸化

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Paucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1-4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S and the DpN1 cells produced lower concentrations of SeAP than the Tn5B1-4 cells. Less than 5% of the glycans attached to SeAP produced by the Tn5B 1-4 cells had complex forms. Glycans attached to SeAP from A7S cells contained 4% hybrid and 8% complex forms. Galactosylated biantennary structures were identified. Glycans attached to SeAP produced by the DpNl cell line had 6% hybrid and 26% complex forms. Of the complex forms in SeAP from DpN1, 13% were identified as sialylated glycans. The galactosyl-transferase activity of the three cell lines was measured and correlated to their ability to produce complex forms. Even though neither novel cell line produced as much recombinant protein as the Tn5B1-4 cells, the glycosylation of SeAP expressed by both cell lines was more complete. These novel cell lines represent interesting alternatives for the production of complex glycosylated proteins utilizing the BEVS.
机译：甘露糖或寡甘露糖结构通常附着在昆虫细胞产生的糖蛋白上，而哺乳动物糖蛋白通常具有复杂的聚糖。尽管它具有高生产率和多功能性，但缺乏复杂的糖基化限制了昆虫细胞杆状病毒表达载体系统（BEVS）的使用。能够进行复杂糖基化的细胞系的可用性可以克服这一问题，并可能增加BEVS的效用。在这项工作中，评估了两种新的细胞系，一种来自Pseudaletia unipuncta（A7S），另一种来自Danaus plexippus（DpN1），产生并糖基化重组蛋白（分泌型人胎盘碱性磷酸酶，SeAP）的能力。 Tn5B1-4细胞以低传代数（<200）产生的SeAP用于比较。确定了DpN1细胞产生SeAP的最佳条件，并定量测定了细胞系产生的SeAP的糖基化谱。 A7S和DpN1细胞均比Tn5B1-4细胞产生更低的SeAP浓度。 Tn5B 1-4细胞产生的与SeAP相连的聚糖中只有不到5％具有复杂形式。从A7S细胞附着到SeAP的聚糖包含4％杂种和8％复杂形式。鉴定了半乳糖基化的双天线结构。 DpN1细胞系产生的附着于SeAP的聚糖具有6％的杂种形式和26％的复杂形式。在来自DpN1的SeAP中的复杂形式中，有13％被鉴定为唾液酸化聚糖。测量了三个细胞系的半乳糖基转移酶活性，并将其与产生复杂形式的能力相关联。尽管没有一种新的细胞系能产生与Tn5B1-4细胞一样多的重组蛋白，但两种细胞系表达的SeAP的糖基化作用都更加完整。这些新颖的细胞系代表了利用BEVS生产复杂糖基化蛋白的有趣替代方法。

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