School of Biology and Biological Engineering, South China University of Technology, 382 East Outer Loop Road, University Park, Guangzhou, 510006, China.
Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
Microb Cell Fact. 2023 Oct 30;22(1):224. doi: 10.1186/s12934-023-02229-5.
Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release.
In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72-97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%.
Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.
蛋白质纯化仍然是生物科学和生物技术的关键需求。它通常需要多轮色谱步骤，既昂贵又耗时。我们的实验室之前报道了一种可切割的自聚集标签（cSAT）方案，用于简化蛋白质表达和纯化。该标签由自组装肽（SAP）和可控自切割内含肽组成。SAP 驱动靶蛋白进入活性聚集体，然后通过内含肽介导的切割，靶蛋白被释放。在这里，我们报告了一种新的 cSAT 方案，其中自组装肽被盐诱导的自组装肽取代。这使得靶蛋白首先以可溶性形式表达，然后添加盐，随后将靶蛋白驱动到聚集形式，然后进行切割和释放。
在这项研究中，我们在 cSAT 方案中使用 MpA（MKQLEDKIEELLSKAAMKQLEDKIEELLSK）作为第二类自组装肽。该方案利用低盐浓度使融合蛋白保持可溶性，同时通过离心去除不溶性细胞物质。然后，盐触发 MpA 介导的融合自组装，去除可溶性背景宿主细胞蛋白。最后，内含肽介导的切割将靶蛋白释放到溶液中。作为概念验证，我们成功纯化了四种蛋白质和肽（人生长激素，22.1 kDa；LCB3，7.7 kDa；SpyCatcherΔN-ELP-SpyCatcherΔN，26.2 kDa；和木聚糖酶，45.3 kDa），产量范围为 12 至 87 mg/L。这与经典的 His 标签法在产量和纯度（72-97%）方面相当，但没有 His 标签。通过使用包括离子交换色谱和尺寸排阻色谱在内的两步柱纯化过程，纯度提高到 99%以上。
我们的结果表明，盐诱导的自组装肽可以作为一种可控的聚集标签，在需要靶蛋白可溶性表达的应用中可能具有优势。这项工作还证明了利用盐诱导的自组装肽进行蛋白质分离的潜力和优势。
