A simple and efficient method for generating high-quality recombinant Mical enzyme for in vitro assays.
A simple and efficient method for generating high-quality recombinant Mical enzyme for in vitro assays.
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DOI：
10.1016/j.pep.2016.05.008
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发表时间：
2016-11
影响因子：
1.6
通讯作者：
Terman JR
中科院分区：
文献类型：
--
作者：
Wu H;Hung RJ;Terman JR
我们最近已经确定了一个新的家庭的多结构域氧化还原酶（氧化还原）酶，MICALs，直接调节肌动蛋白细胞骨架元素所需的形态，运动，和轨迹的细胞。我们的遗传分析表明，Mical是必要的和足够的肌动蛋白组织和细胞的影响，在体内和我们的生化分析与纯化的Mical蛋白揭示，Mical利用其氧化还原活性，直接拆卸肌动蛋白丝。这些结果确定Mical蛋白作为新的肌动蛋白分解因子，并揭示了直接调节肌动蛋白细胞骨架的氧化还原信号机制。这些结果也为深入表征Mical酶奠定了基础。然而，很难获得足够量的高纯度Mical蛋白，以进行进一步的生化，结构，成像，催化和其他高精度研究。在此，我们描述了在细菌中表达高水平的可溶性重组Mical蛋白的方法。同样，我们设计了一种新的纯化策略，能够快速有效地纯化毫克量的高纯度和>99%活性Mical蛋白。这种产生大量高纯度和活性Mical蛋白的新策略将有助于旨在表征Mical的生化学，酶学和结构生物学及其对肌动蛋白丝动力学的影响的研究目标。
We have recently identified a new family of multidomain oxidoreductase (redox) enzymes, the MICALs, that directly regulate the actin cytoskeletal elements necessary for the morphology, motility, and trajectory of cells. Our genetic assays reveal that Mical is both necessary and sufficient for actin organization and cellular effects in vivo and our biochemical assays with purified Mical protein reveal that Mical utilizes its redox activity to directly disassemble actin filaments. These results identify Mical proteins as novel actin disassembly factors and uncover a redox signaling mechanism that directly regulates the actin cytoskeleton. These results have also set the stage for in-depth characterization of the Mical enzyme. However, it has been difficult to obtain sufficient amounts of highly-pure Mical protein to conduct further biochemical, structural, imaging, catalytic, and other high-precision studies. Herein, we describe a means for expressing high levels of soluble recombinant Mical protein in bacteria. Likewise, we have designed a new purification strategy that enables the rapid and efficient purification of milligram quantities of highly-pure and >99% active Mical protein. This new strategy for generating large amounts of highly-pure and active Mical protein will aid research objectives designed to characterize the biochemical, enzymology, and structural biology of Mical and its effects on actin filament dynamics.