A unified purification method for actin-binding proteins using a TEV-cleavable His-Strep-tag

IF 1.6 Q2 MULTIDISCIPLINARY SCIENCES MethodsX Pub Date : 2024-08-06 DOI:10.1016/j.mex.2024.102884

Daichi Nakajima , Nozomi Takahashi , Takanari Inoue , Shin-ichiro M. Nomura , Hideaki T. Matsubayashi

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Abstract

The actin cytoskeleton governs the dynamic functions of cells, ranging from motility to phagocytosis and cell division. To elucidate the molecular mechanism, in vitro reconstructions of the actin cytoskeleton and its force generation process have played essential roles, highlighting the importance of efficient purification methods for actin-binding proteins. In this study, we introduce a unified purification method for actin-binding proteins, including capping protein (CP), cofilin, ADF, profilin, fascin, and VASP, key regulators in force generation of the actin cytoskeleton. Exploiting a His-Strep-tag combined with a TEV protease cleavage site, we purified these diverse actin-binding proteins through a simple two-column purification process: initial purification through a Strep-Tactin column and subsequent tag removal through the reverse purification by a Ni-NTA column. Biochemical and microscopic assays validated the functionality of the purified proteins, demonstrating the versatility of the approach. Our methods not only delineate critical steps for the efficient preparation of actin-binding proteins but also hold the potential to advance investigations of mutants, isoforms, various source species, and engineered proteins involved in actin cytoskeletal dynamics.

•
Unified purification method for various actin-binding proteins.
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His-Strep-tag and TEV protease cleavage for efficient purification.
•
Functional validation through biochemical and microscopic assays.

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使用 TEV 可切除 His-Strep 标记的肌动蛋白结合蛋白的统一纯化方法

肌动蛋白细胞骨架支配着细胞的动态功能，包括运动、吞噬和细胞分裂。为了阐明其分子机制，体外重建肌动蛋白细胞骨架及其受力过程发挥了至关重要的作用，这凸显了高效纯化肌动蛋白结合蛋白方法的重要性。在这项研究中，我们介绍了一种统一的肌动蛋白结合蛋白纯化方法，其中包括盖层蛋白（CP）、cofilin、ADF、profilin、fascin 和 VASP，它们都是肌动蛋白细胞骨架力发生过程中的关键调控因子。我们利用 His-Strep 标记和 TEV 蛋白酶裂解位点，通过简单的双柱纯化过程纯化了这些不同的肌动蛋白结合蛋白：通过 Strep-Tactin 柱进行初步纯化，然后通过 Ni-NTA 柱进行反向纯化去除标记。生化和显微检测验证了纯化蛋白的功能，证明了这种方法的多功能性。我们的方法不仅划定了高效制备肌动蛋白结合蛋白的关键步骤，而且有望推动对突变体、异构体、各种来源物种以及参与肌动蛋白细胞骨架动力学的工程蛋白的研究。

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