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Related Concept Videos

Oligosaccharide Assembly01:24

Oligosaccharide Assembly

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Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
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Related Experiment Video

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Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry
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Immobilized exoglycosidase matrix mediated solid phase glycan sequencing.

Róbert Farsang1, Noémi Kovács2, Márton Szigeti1

  • 1Translational Glycomics Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary.

Analytica Chimica Acta
|June 9, 2022
PubMed
Summary
This summary is machine-generated.

Researchers developed three immobilized exoglycosidases for robust N-glycan sequencing. This method enhances glycoprotein analysis for biopharmaceutical and biomedical applications, improving diagnostic and therapeutic strategies.

Keywords:
6HIS-tagged enzyme productionCapillary electrophoresisExoglycosidasesGlycan sequencingImmobilization

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Area of Science:

  • Biochemistry
  • Glycobiology
  • Biotechnology

Background:

  • Characterizing glycoprotein glycans is crucial for biopharmaceutical development and biomedical research.
  • Existing methods for glycan analysis can be time-consuming and lack robustness.
  • Efficient tools are needed to support rapid and reliable N-glycan sequencing.

Purpose of the Study:

  • To design and produce immobilized exoglycosidases for enhanced solid-phase N-glycan sequencing.
  • To create a robust and efficient enzymatic system for analyzing complex carbohydrate structures.
  • To support the biopharmaceutical industry and biomedical field with advanced glycan analysis tools.

Main Methods:

  • Production of three 6HIS-tagged exoglycosidases (neuraminidase, β-galactosidase, hexosaminidase) in bacterial expression systems.
  • Oriented immobilization of enzymes via the 6HIS-tag for improved active site accessibility.
  • Premixing enzymes in a matrix format and processing in a low-salt buffer for stability and storage.
  • Demonstration of digestion efficiency using solid-phase sequencing and capillary electrophoresis.

Main Results:

  • High yields of bacterial expression for all three exoglycosidases.
  • Successful oriented immobilization leading to high digestion performance.
  • Demonstrated digestion efficiencies on a glycoprotein therapeutic (palivizumab) and human serum glycans.
  • Validation of the immobilized enzyme system for rapid N-glycan sequencing.

Conclusions:

  • The developed immobilized exoglycosidase system offers a robust and efficient method for N-glycan sequencing.
  • This enzymatic approach facilitates rapid characterization of carbohydrate moieties in glycoproteins.
  • The technology has significant implications for quality control in biopharmaceutical manufacturing and biomarker discovery.