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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
Author(s) -
Mokrzycki-Issartel Nathalie,
Bouchon Bernadette,
Farrer Sibille,
Berland Patricia,
Laparra Hélène,
Madelmont Jean-Claude,
Theisen Manfred
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)00916-5
Subject(s) - glycosylation , glycan , chemistry , glycoprotein , recombinant dna , biochemistry , n linked glycosylation , time of flight mass spectrometry , matrix assisted laser desorption/ionization , trypsin , chromatography , enzyme , desorption , gene , ion , organic chemistry , adsorption , ionization
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.