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首页> 外文期刊>BMC Biotechnology >One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli
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One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli

机译：一种在大肠杆菌中生产天然且与Tat融合的重组人α-突触核蛋白的单一方法

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Background Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. Results A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. Conclusions To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson’s disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.
机译：背景技术人类α-突触核蛋白是一种小而天然的未折叠蛋白，其原纤维形式是路易氏体的主要成分，路易氏体是帕金森氏病的病理标志。实验证据表明，α-突触核蛋白的聚集是触发神经毒性的关键事件，尽管其他发现也提出了α-突触核蛋白对氧化应激的保护作用。解决这种保护作用机制的一种方法是通过使用与HIV Tat的Tat转化域融合的嵌合蛋白（称为TAT-α-synuclein）将这种蛋白传递到细胞内来评估α-突触核蛋白介导的保护作用。结果设计了一种可靠的方案，可以有效表达和纯化两种不同形式的人α-突触核蛋白。编码天然α-突触核蛋白的合成cDNA和具有来自HIV的Tat蛋白转导域的融合蛋白在BL21（DE3）大肠杆菌菌株中过表达为His-tagged蛋白。重组蛋白主要定位在周质间隙（≥85％）。通过使用快速纯化方案，基于周质空间含量的回收和金属螯合色谱法，重组α-突触核蛋白蛋白形式可以一步纯化至纯度≥95％。两种α-突触核蛋白重组蛋白均形成原纤维，而TAT-α-突触核蛋白在微摩尔浓度范围内也具有细胞毒性。结论为进一步表征α-突触核蛋白在体外和体内的神经毒性的分子机制，并评估细胞外α-突触核蛋白与帕金森氏病的发病机理和进展的相关性，是一种产生不同高质量形式的这种病理学方法的合适方法。蛋白质是必需的。我们优化的表达和纯化程序提供了一种生产可溶性重组α-突触核蛋白的不同形式（即天然和TAT融合形式）的简便，快捷的方法，比以前描述的程序更容易。

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