A transient tobacco expression system coupled to MALDI-TOF-MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants
| Autor: | Bernadette Bouchon, Jean Claude Madelmont, Hélène Laparra, Manfred Theisen, Sibille Farrer, Nathalie Mokrzycki-Issartel, Patricia Berland |
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| Rok vydání: | 2003 |
| Předmět: |
N-linked glycosylation
Glycan Glycosylation Molecular Sequence Data Biophysics Gene Expression In Vitro Techniques Biochemistry law.invention chemistry.chemical_compound Dogs Structural Biology law Polysaccharides Tobacco Genetics Animals Gastric lipase Lipase Molecular Biology Glycoproteins chemistry.chemical_classification Differential targeting Binding Sites biology Molecular Structure Transient expression Cell Biology Plants Genetically Modified MALDI-TOF-MS Recombinant therapeutic proteins from plants Recombinant Proteins carbohydrates (lipids) Matrix-assisted laser desorption/ionization chemistry Carbohydrate Sequence Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Recombinant DNA biology.protein Glycoprotein |
| Zdroj: | FEBS letters. 552(2-3) |
| ISSN: | 0014-5793 |
| Popis: | Tobacco-based transient expression was employed to elucidate the impact of differential targeting to subcellular compartments on activity and quality of gastric lipase as a model for the production of recombinant glycoproteins in plants. Overall N-linked glycan structures of recombinant lipase were analyzed and for the first time sugar structures of its four individual N-glycosylation sites were determined in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on a trypsin digest without isolation or deglycosylation of the peptides. Three glycosylation sites contain both complex-type N-glycans and high-mannose-type structures, the fourth is exclusively linked to high-mannose glycans. Although the overall pattern of glycan structures is influenced by the targeting, our results show that the type of glycans found linked to a given Asn residue is largely influenced by the physico-chemical environment of the site. The transient tobacco system combined with MALDI-TOF-MS appears to be a useful tool for the evaluation of glycoprotein production in plants. |
| Databáze: | OpenAIRE |
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