Production and N-glycosylation of recombinant human cell adhesion molecule L1 from insect cells using the stable expression system. Effect of dimethyl sulfoxide
| Autor: | Harald S. Conradt, Ricardo M. Gouveia, Júlia Costa, Sebastian Kandzia |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Glycan
Glycosylation Bioengineering Sf9 Neural Cell Adhesion Molecule L1 Spodoptera Protein Engineering Transfection Applied Microbiology and Biotechnology Fucose 03 medical and health sciences chemistry.chemical_compound N-linked glycosylation Animals Humans Dimethyl Sulfoxide Cloning Molecular Cells Cultured 030304 developmental biology 0303 health sciences biology Dimethyl sulfoxide Cell adhesion molecule 030302 biochemistry & molecular biology General Medicine Molecular biology Recombinant Proteins Genetic Enhancement Biochemistry chemistry Cell culture biology.protein Biotechnology |
| Zdroj: | Journal of biotechnology. 145(2) |
| ISSN: | 1873-4863 |
| Popis: | L1 is a cell adhesion molecule that is heavily glycosylated and is essential for normal development of the central nervous system. In this work, we compare the N -glycosylation of the L1 mutant that consists of immunoglobulin domains 5 and 6 (L1/Ig5–6), expressed in insect Spodoptera frugiperda Sf9 and Trichoplusia ni Tn cells, using the stable expression system. L1/Ig5–6 levels of 30 and 8 mg l −1 were obtained from the two cell lines, respectively. The N -glycans were characterized by high-performance anion-exchange-chromatography with pulsed-amperometric-detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N -glycans from Sf9 cells were more homogeneous and consisted predominantly of the paucimannose-type structure Manα6(Manα3)Manβ4GlcNAcβ4(Fucα6)GlcNAc. On the other hand, the N -glycans from Tn cells were more heterogeneous and consisted of paucimannose-type structures with or without terminal N -acetylglucosamine. Allergenic proximal α3-linked fucose was only found in Tn cells. Dimethyl sulfoxide at 1.5% concentration has been found to increase the levels of L1/Ig5–6 and the L1 ectodomain in the Sf9 and Tn cells, without affecting cell viability nor protein integrity. Furthermore, the N -glycan composition of L1/Ig5–6 was not affected by dimethyl sulfoxide, with only a slight increase in the percentage of the minor high-mannose-type structures. |
| Databáze: | OpenAIRE |
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