Re-visiting the endogenous capacity for recombinant glycoprotein sialylation by baculovirus-infected Tn-4h and DpN1 cells
| Autor: | Alexander Hillar, Donald L. Jarvis |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Baculoviridae
Sialoglycoproteins Genetic Vectors Immunoblotting Sf9 Spodoptera Biochemistry Chromatography Affinity Culture Media Serum-Free law.invention law Sialoglycoprotein Polysaccharides Lectins Animals Glutathione Transferase chemistry.chemical_classification biology biology.organism_classification Alkaline Phosphatase Fetuin Molecular biology Recombinant Proteins carbohydrates (lipids) Lepidoptera chemistry Cell culture Recombinant DNA biology.protein Cattle Original Article Glycoprotein Fetal bovine serum |
| Zdroj: | Glycobiology. 20(10) |
| ISSN: | 1460-2423 |
| Popis: | It was previously reported that Tn-4h and DpN1 cells have the endogenous capacity to efficiently sialylate secreted alkaline phosphatase (SEAP) when infected with a baculovirus expression vector. In contrast, it has been found that lepidopteran insect cell lines that are more widely used as hosts for baculovirus vectors typically fail to sialylate SEAP and other recombinant glycoproteins. Thus, the N-glycan processing capabilities of Tn-4h and DpN1 cells are of potential interest to investigators using the baculovirus expression system for recombinant glycoprotein production. In this study, we experimentally re-assessed the ability of Tn-4h and DpN1 cells to sialylate SEAP with Sf9 and glyco-engineered Sf9 cells (SfSWT-1) as negative and positive controls, respectively. Our results showed that the SEAP purified from SfSWT-1 cells was strongly sialylated and initially indicated that the SEAP purified from Tn-4h cells was weakly sialylated. However, further analyses suggested that the SEAP produced by Tn-4h cells only appeared to be sialylated because it was contaminated with an electrophoretically indistinguishable sialoglycoprotein derived from fetal bovine serum. We subsequently expressed, purified, and analyzed a second recombinant glycoprotein (GST-SfManI) from all four cell lines and found that only the SfSWT-1 cells were able to detectably sialylate this product. Together, these results showed that neither Tn-4h nor DpN1 cells efficiently sialylated SEAP or GST-SfManI when infected by baculovirus expression vectors. Furthermore, they suggested that previous reports of efficient SEAP sialylation by Tn-4h and DpN1 cells probably reflect contamination with a sialylated, co-migrating glycoprotein, perhaps bovine fetuin, derived from the serum used in the insect cell growth medium. |
| Databáze: | OpenAIRE |
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