Immobilized exoglycosidase matrix mediated solid phase glycan sequencing

Full characterization of the attached carbohydrate moieties of glycoproteins is of high importance for both the rapidly growing biopharmaceutical industry and the biomedical field. In this paper we report the design and production of three important 6HIS-tagged exoglycosidases (neuraminidase, beta-galactosidase and hexosaminidase) to support rapid solid phase N-glycan sequencing with high robustness using immobilized enzymes. The exoglycosidases were generated in bacterial expression systems with high yield. Oriented immobilization via the 6HIS-tag portion of the molecules supported easy accessibility to the active sites and consequently high digestion performance. The three exoglycosidases were premixed in an appropriate matrix format and processed in a low-salt buffer to support long term storage. The digestion efficiencies of the immobilized enzymes were demonstrated by using solid phase sequencing in conjunction with capillary electrophoresis analysis of the products on a commercial glycoprotein therapeutic (palivizumab) and human serum derived fluorophore labeled glycans.

Automated summary

This paper reports the design and production of 6HIS-tagged exoglycosidases for efficient solid phase N-glycan sequencing. The enzymes were generated in bacterial expression systems and immobilized via the 6HIS-tag for high digestion performance. The immobilized enzymes were demonstrated to have high efficiency in digesting glycoproteins through analysis of commercial glycoprotein therapeutic and fluorophore labeled glycans in human serum.