Expression and immobilization of novel N-glycan-binding protein for highly efficient purification and enrichment of N-glycans, N-glycopeptides, and N-glycoproteins.

Anal Bioanal Chem

Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics - Hubei Bioinformatics & Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China.

Published: December 2024

AI Article Synopsis

  • Comprehensive enrichment of N-glycans and associated molecules is crucial in N-glycomics research to simplify samples, reduce impurities, and enhance analysis quality.
  • The research focuses on a specific Fbs1 GYR variant (Fg), which has a strong binding affinity for the core pentasaccharide of N-glycans, making it effective for this purpose.
  • The study involved cloning the Fg into a GST-tagged vector for easy purification, resulting in a stable and effective protein that can efficiently isolate N-glycans and their derivatives for analytical applications.

Article Abstract

Comprehensive and selective enrichment of N-glycans, N-glycopeptides, and N-glycoproteins prior to analysis is of great significance in N-glycomics research, reducing sample complexity, removing impurity interference, increasing sample abundance and enhancing signal intensity. However, only an Fbs1 (F-box protein that recognizes sugar chain 1) GYR variant (Fg) can enrich these N-glycomolecules solely due to its substantial binding affinity for the core pentasaccharide motif of N-glycans. Stationary phase separation is commonly used to enrich N-glycomolecules efficiently. Herein, DNA encoding the Fg was cloned into pGEX-4T-1, and the protein was expressed with a GST tag, which facilitates the convenient and efficient immobilization of recombinant GST-tagged Fg to GSH agarose resin. The yield of the GST-tagged Fg reached to 0.05 g/L after optimization of the induction condition, and the purified protein exhibited good identification ability and excellent stability for months. In particular, the immobilized GST-tagged Fg can enrich N-glycans released by PNGase F and capture derivatized N-glycans possessing an intact terminal N-acetyl glucosamine (GlcNAc). Validation of immobilized GST-tagged Fg with standard N-glycopeptides and N-glycoproteins revealed its high loading capacity, sensitivity, and selectivity. The novel immobilized GST-tagged Fg is a convenient and efficient enrichment material specific for N-glycans, N-glycopeptides, and N-glycoproteins, suggesting excellent performance and prospects for industrial application.

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Source
http://dx.doi.org/10.1007/s00216-024-05583-4DOI Listing

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