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High yield purification and first structural characterization of the full-length bacterial toxin CNF1

机译：高产纯化和全长细菌毒素CNF1的第一结构表征

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The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15 degrees C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018
机译：细胞毒性坏死因子1（CNF1）是由某些大肠杆菌菌株分泌的细菌毒素，其引起严重病理学，使其成为毒理学的关键兴趣蛋白质。并行地，影响重要神经元过程的CNF1能力，如神经元植物，星形织物支持和高效的ATP生产，以有效地用于治疗神经系统疾病，使其成为治疗的有希望的候选者。尽管如此，关于CNF1的动作和结构机制仍有一些未解决的问题可能是由于难以实现足够量的全长蛋白质进行进一步研究而导致的。在这里，我们提出了一种有效的策略，用于从大肠杆菌提取物（CNF1-H8）中作为其标记的重组蛋白的这种毒素的生产和纯化。 CNF1-H8在15摄氏度的低温下表达，以减少其特征性降解。然后，使用固定化的金属亲和色谱法（IMAC）和尺寸排阻色谱法实现其纯化，以便以高纯度的形式收集每升培养物的高达8mg蛋白质。常规动态光散射（DLS）实验表明，重组蛋白制剂均匀并保持该状态长时间。此外，通过在纯化的RHOA和HEP-2培养细胞上测试其活性来确认CNF1-H8官能度。最后，通过圆形二色性（CD）进行次级结构和热稳定性方面的全长毒素的第一结构表征。这些研究表明，我们的系统可用于生产高量的纯重组蛋白，以进行详细的结构分析。 （c）2017美国化学工程师生物科技学院。 Prog。，34：150-159,2018

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