Farsang Róbert, Kovács Noémi, Szigeti Márton, Jankovics Hajnalka, Vonderviszt Ferenc, Guttman András
Translational Glycomics Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary.
Bio-Nanosystems Laboratory, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary.
Anal Chim Acta. 2022 Jul 4;1215:339906. doi: 10.1016/j.aca.2022.339906. Epub 2022 May 14.
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, β-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.
对糖蛋白上连接的碳水化合物部分进行全面表征，对快速发展的生物制药行业和生物医学领域都至关重要。在本文中，我们报告了三种重要的带有6组氨酸标签的外切糖苷酶（神经氨酸酶、β-半乳糖苷酶和己糖胺酶）的设计与生产，以支持使用固定化酶进行具有高稳健性的快速固相N-聚糖测序。这些外切糖苷酶在细菌表达系统中高产生成。通过分子的6组氨酸标签部分进行定向固定，有助于活性位点的轻松可达性，从而实现高消化性能。这三种外切糖苷酶以适当的基质形式预混合，并在低盐缓冲液中处理以支持长期储存。通过在商业糖蛋白治疗药物（帕利珠单抗）和人血清衍生的荧光团标记聚糖上结合毛细管电泳分析进行固相测序，证明了固定化酶的消化效率。
