Evert Ben, Vezina Ben, Rehm Bernd H A
Centre for Cell Factories and Biopolymers, Griffith Institute for Drug Discovery, Griffith University, Brisbane 4111, Australia.
ACS Appl Bio Mater. 2020 Dec 21;3(12):8911-8922. doi: 10.1021/acsabm.0c01195. Epub 2020 Nov 24.
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 . 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.
常见的蛋白质纯化方法依赖于目标蛋白通过融合标签与树脂的非共价亲和结合，这种方法存在局限性，例如在去除杂质过程中目标蛋白会泄漏、限制洗涤条件的严格性，并且通常需要在纯化后去除标签。在此，我们通过生物工程构建了一种一次性使用的树脂，开发了捕获、保留和释放（C-H-R）方法，该树脂由共展示两个SpyCatcher（SpyC）结构域和分选酶A（SrtA）的聚羟基丁酸酯珠组成。这种核壳树脂是通过工程化组装而成的。带有SrtA切割位点的SpyTagged（SpyT）靶标在SpyT后被特异性连接到SpyC上，而可诱导的SrtA催化释放出无标签的纯目标蛋白。SpyT仍共价结合在树脂上。从复杂混合物中分离出了三种不同的蛋白质，纯度和产量都很高。C-H-R生物分离方法使用了一种创新的树脂，该树脂与目标蛋白形成共价连接，并使用可诱导的近端SrtA作为蛋白质纯化的概念。
