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Glycobiology_Course_lesson_4

Glycobiology_Course_lesson_4 - Glycobiology Course IIIa...

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Glycobiology Session 3, slide 1 Glycobiology Course IIIa Producing glycoproteins. Roland Contreras Nico Callewaert Wouter Laroy
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Glycobiology Session 3, slide 2 Expression hosts: cultured mammalian cells.
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Glycobiology Session 3, slide 3 CMP-NANA hydroxylase
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Glycobiology Session 3, slide 4 Expression hosts: cultured mammalian cells with a defect in the glycosylation pathway. Example: Lec1.
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Glycobiology Session 3, slide 5 Expression hosts: insect cells.
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Glycobiology Session 3, slide 6 Expression hosts: yeasts and fungi, particularly Pichia pastoris and Aspergillus niger
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Glycobiology Session 3, slide 7 Expression hosts: yeasts and fungi, particularly Pichia pastoris and Aspergillus niger Engineering in Pichia and in Aspergillus is a very active cutting- edge field of biotechnology at this moment. Breakthroughs have been achieved that bring production of human biopharmaceuticals within reach.
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Glycobiology Session 3, slide 8 Expression hosts: plants.
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Glycobiology Session 3, slide 9 Glycobiology Course IIIb Analytical techniques. Roland Contreras Nico Callewaert Wouter Laroy
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Glycobiology Session 3, slide 10 Case study: a pharmaceutical company has produced a new monoclonal antibody for therapeutic purposes. You are asked to analyse the glycosylation of this purified antibody. For the initial study, you will receive a large amount of the molecule (1 mg). You have to find out whether the molecule is glycosylated, what type of sugars are present and what structures are present. Moreover, you are required to provide a relative abundance of these structures, and report back whether there are any known immunogenic substituents present. After approval of the antibody drug, The Food and Drug administration requires that each batch of the antibody that will be put on the market be checked for consistency, and the company needs a reliable method with high throughput and high sensitivity to be used in their routine quality control laboratory. This method should compare the glycosylation of each batch with the ‘reference sample’ of which you analysed the structures in detail before.
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Glycobiology Session 3, slide 11 Analytical techniques: detection (are there sugars on the protein?) Carbohydrate-specific stains: Several commercial kits for glycoprotein detection (PAGE/Western): GelCode Glycoprotein Staining (Pierce) Pro-Q Emerald (Molecular Probes) ECL Glycoprotein detection (Amersham-Pharmacia) DIG Glycan Detection (Roche) Glyco-Pro kit (Sigma) – … Total protein stain Glycoprotein stain All kits based on the oxidation of vicinal diols, followed by reaction with a chromophore, fluorophore, or antigen molecule for subsequent detection by color, fluorescence, or antibody-based assay. P.S. you can make some of the stuff cheaper from commonly available reagents.
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Glycobiology Session 3, slide 12 Analytical techniques: what type of sugars?
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  • Spring '10
  • JohanGrooten
  • Glycoprotein, Glycobiology Session, Roland Contreras Nico Callewaert, chromatography Structural analysis

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