新型 N-糖结合蛋白的表达和固定,用于高效纯化和富集 N-糖、N-糖肽和 N-糖蛋白。

IF 3.8 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Analytical and Bioanalytical Chemistry Pub Date : 2024-12-01 Epub Date: 2024-10-16 DOI:10.1007/s00216-024-05583-4
Liang Zhang, Wenhui Wang, Yueqin Yang, Pengjie Li, Xiang Liu, Wenjie Zhu, Wei Yang, Song Wang, Yawei Lin, Xin Liu
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引用次数: 0

摘要

在分析前对 N-聚糖、N-聚糖肽和 N-聚糖蛋白进行全面和选择性富集对 N-聚糖研究意义重大,可降低样品复杂性、去除杂质干扰、提高样品丰度并增强信号强度。然而,只有 Fbs1(识别糖链 1 的 F-box 蛋白)GYR 变体(Fg)能富集这些 N-糖分子,这完全是因为它与 N-糖的核心五糖基团有很强的结合亲和力。固定相分离通常用于高效富集 N-糖分子。本文将编码Fg的DNA克隆到pGEX-4T-1中,并用GST标签表达该蛋白,从而方便高效地将重组的GST标签Fg固定到GSH琼脂糖树脂上。优化诱导条件后,GST 标记 Fg 的产率达到 0.05 g/L,纯化后的蛋白具有良好的鉴定能力和数月的稳定性。其中,固定化 GST 标记 Fg 能富集 PNGase F 释放的 N-聚糖,并捕获具有完整末端 N-乙酰葡糖胺(GlcNAc)的衍生化 N-聚糖。用标准 N-糖肽和 N-糖蛋白对固定化 GST 标记 Fg 进行验证后发现,它具有很高的负载能力、灵敏度和选择性。新型固定化 GST 标记 Fg 是一种方便、高效的 N-聚糖、N-糖肽和 N-糖蛋白特异性富集材料,具有优异的性能和工业应用前景。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Expression and immobilization of novel N-glycan-binding protein for highly efficient purification and enrichment of N-glycans, N-glycopeptides, and N-glycoproteins.

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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来源期刊
CiteScore
8.00
自引率
4.70%
发文量
638
审稿时长
2.1 months
期刊介绍: Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.
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