Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase
Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase
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DOI：
10.1002/biot.201500030
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发表时间：
2016-02-01
影响因子：
4.7
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
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作者：
Li, Jian;Lawton, Thomas J.;Jewett, Michael C.
多铜氧化酶（MCOs）广泛分布于所有生物领域，并参与多种重要的氧化反应。这些酶具有潜在的生物技术应用；然而，传统重组宿主的低表达率、溶解度问题和铜辅助因子组装不良阻碍了其应用。作为传统重组蛋白表达的替代方案，我们展示了使用无细胞蛋白合成（CFPS）产生高可溶性滴度的复杂MCO蛋白的能力。具体来说，我们报道了在大肠杆菌为基础的无细胞转录-翻译系统中MCOs的产生。经过20小时的间歇反应，总产率高达1.2 mg mL（-1）。95%以上的蛋白质是可溶的，通过简单的cfps后添加铜离子以CuSO4的形式获得活性。放大反应可在15 ~ 100 μ L范围内进行，而生产效率和溶解度均未降低。CFPS滴度高于体内表达滴度，且更易溶解，避免了包涵体的形成。我们的工作将无细胞平台的效用扩展到生产含有铜辅助因子的活性蛋白，并展示了一种生产MCOs的简单方法。
Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli-based cell-free transcription-translation system. Total yields as high as 1.2 mg mL(-1) were observed after a 20-h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post-CFPS addition of copper ions in the form of CuSO4. Scale-up reactions were achieved from 15 to 100 mu L without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell-free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs.