Common protein purification processes rely on noncovalent affinity binding of the target protein to resin via fusion tags, which has limitations such as leakage of target during the removal of impurities, restricting stringency of wash conditions, and often requires postpurification removal of tags. Here, we developed the Catch, Hold, and Release (C-H-R) method by bioengineering a single-use resin composed of polyhydroxybutyrate beads codisplaying two SpyCatcher (SpyC) domains and Sortase A (SrtA). This core–shell resin was assembled by engineered Escherichia coli. SpyTagged (SpyT) targets with a SrtA cleavage site after SpyT were specifically ligated to SpyC, while inducible SrtA catalyzed the release of the pure tagless target. SpyT remained covalently bound to the resin. Three diverse proteins were isolated from complex mixtures with high purity and yields. The C-H-R bioseparation method uses an innovative resin engaging a covalent link to the target and an inducible proximal SrtA as a protein purification concept.

中文翻译：

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利用共价中间体进行无标签蛋白纯化的催化活性生物分离树脂

常见的蛋白质纯化过程依赖于目标蛋白通过融合标签与树脂的非共价亲和结合，其存在局限性，例如在去除杂质期间目标蛋白的泄漏，限制洗涤条件的严格性，并且通常需要在纯化后去除标签。在这里，我们通过生物工程开发了一种一次性树脂，该树脂由共同展示两个 SpyCatcher (SpyC) 结构域和 Sortase A (SrtA) 的聚羟基丁酸酯珠组成，从而开发了捕获、保持和释放 (CHR) 方法。这种核壳树脂由工程大肠杆菌组装而成. 在 SpyT 后具有 SrtA 切割位点的 SpyTagged (SpyT) 靶标与 SpyC 特异性连接，而诱导型 SrtA 催化纯无标记靶标的释放。SpyT 仍然与树脂共价结合。从高纯度和高产率的复杂混合物中分离出三种不同的蛋白质。CHR 生物分离方法使用一种创新的树脂，该树脂与靶标共价连接，并使用可诱导的近端 SrtA 作为蛋白质纯化概念。