The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.

中文翻译：

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一种基于SUMO融合重组蛋白和Ulp1蛋白酶的细胞表面展示的简单快速的蛋白纯化方法。

由于包括亲和纯化，凝胶过滤和离子交换色谱在内的常规纯化方法需要复杂的操作步骤且成本高昂，因此开发高效蛋白质纯化的新方法仍然是生物技术领域的研究重点。在这里，我们描述了一种简单而快速的蛋白质纯化策略，其中SUMO标签和Ulp1蛋白酶分别在大肠杆菌细胞上表面展示。蛋白质诱导后，收获细胞，重悬于切割缓冲液中，并一起孵育以进行切割。在这种方法中，表面展示的Ulp1裂解了膜锚定的SUMO融合蛋白，导致目标蛋白从SUMO的C端释放到溶液中。表面上带有SUMO和Ulp1的细菌细胞可以通过离心轻松去除。为了评估纯化方法，我们使用了红色荧光蛋白（mCherry）。孵育仅30分钟后，即可获得纯化的mCherry蛋白（从1 L细菌培养物中获得7.72±1.05 mg）。蛋白质纯度高于80％，可以通过简单的超滤进一步提高（> 90％）。这项研究为纯化天然形式的重组蛋白提供了一种有希望且简单的策略，该工艺仅需要切割和离心步骤即可。并可以通过简单的超滤进一步改善（> 90％）。这项研究为纯化天然形式的重组蛋白提供了一种有希望且简单的策略，该工艺仅需要切割和离心步骤即可。并可以通过简单的超滤进一步改善（> 90％）。这项研究为纯化天然形式的重组蛋白提供了一种有希望且简单的策略，该工艺仅需要裂解和离心步骤即可。