Yang Xiaofeng, Lin Zhanglin, Xiang Ya, Chen Binrui, Lao Zisha
School of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong, China.
Bio Protoc. 2025 Apr 20;15(8):e5270. doi: 10.21769/BioProtoc.5270.
Protein purification is a critical step in both life sciences and biomanufacturing. Traditional affinity chromatography (AC) methods, including His-tag-based purification, provide high-purity proteins but are limited by the high cost of resins and the need for additional tag-removal steps. In this protocol, we present a reusable SpyDock-modified epoxy resin coupled with a pH-inducible self-cleaving intein for direct purification of proteins with authentic N-termini. This method enables efficient protein purification from cell lysates, achieving high purity (>90%) and yields comparable to the His-tag approach, without requiring tag removal. The SpyDock-modified resin protocol is robust, easy to implement, and cost-effective, making it suitable for both research and large-scale industrial applications. Key features • This protocol offers a robust and straightforward method for purifying proteins with authentic N-termini, eliminating the need for additional tag removal steps. • The approach achieves higher purity and comparable yields to the commercial His-tag method. • The SpyDock-modified epoxy resin is easy to prepare, cost-effective, and reusable.
蛋白质纯化是生命科学和生物制造中的关键步骤。传统的亲和色谱（AC）方法，包括基于His标签的纯化，可提供高纯度蛋白质，但受到树脂成本高以及需要额外的标签去除步骤的限制。在本方案中，我们展示了一种可重复使用的SpyDock修饰环氧树脂，它与pH诱导的自切割内含肽相结合，用于直接纯化具有天然N端的蛋白质。该方法能够从细胞裂解物中高效纯化蛋白质，实现高纯度（>90%），产量与His标签方法相当，且无需去除标签。SpyDock修饰树脂方案稳健、易于实施且具有成本效益，适用于研究和大规模工业应用。关键特性 • 本方案提供了一种稳健且直接的方法来纯化具有天然N端的蛋白质，无需额外的标签去除步骤。 • 该方法可实现更高的纯度，产量与商业His标签方法相当。 • SpyDock修饰的环氧树脂易于制备、具有成本效益且可重复使用。
