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首页> 外文期刊>Biotechnology Progress >Engineering a Recyclable Elastin-like Polypeptide Capturing Scaffold for Non Chromatographic Protein Purification
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Engineering a Recyclable Elastin-like Polypeptide Capturing Scaffold for Non Chromatographic Protein Purification

机译：工程设计可回收的类似弹性蛋白的多肽捕获支架，用于非色谱蛋白的纯化

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摘要

Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium ther-mocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification.
机译：以前，我们报道了一种非色谱蛋白质纯化方法，该方法利用了来自梭状芽孢杆菌的dockerin和cohesin域之间的高度特异性相互作用以及弹性蛋白样多肽（ELP）的可逆聚集特性，从而提供了快速且经济高效的蛋白质纯化方法。然而，由于高结合亲和力，结合的dockerin-intein标签无法从ELP-cohesin捕获支架上完全解离，导致一次性使用。为了通过回收ELP捕获支架来进一步降低纯化成本，采用了钙协调功能部分受损的截短的dockerin结构域。我们证明了截短的dockerin结构域足以充当有效的亲和标记，并且在单个结合步骤中随后从内含肽切割后直接从细胞提取物中纯化了目标蛋白。实现了有效的EDTA介导的结合的dockerin-intein标签与ELP-粘蛋白捕获支架的有效解离，并且将再生的ELP捕获支架重新用于另一个纯化循环，而不会降低纯化效率。这种可回收的基于非色谱的亲和方法为有效且经济高效的蛋白质纯化提供了一种有吸引力的方法。

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