Inclusion bodies (IBs) are biologically active protein aggregates forming natural nanoparticles with a high stability and a slow-release behavior. Because of their nature, IBs have been explored to be used as biocatalysts, in tissue engineering, and also for human and animal therapies. To improve the production and biological efficiency of this nanomaterial, a wide range of aggregation tags have been evaluated. However, so far, the presence in the IBs of bacterial impurities such as lipids and other proteins coexisting with the recombinant product has been poorly studied. These impurities could strongly limit the potential of IB applications, being necessary to control the composition of these bacterial nanoparticles. Thus, we have explored the use of leucine zippers as alternative tags to promote not only aggregation but also the generation of a new type of IB-like protein nanoparticles with improved physicochemical properties. Three different protein constructs, named GFP, J-GFP-F and J/F-GFP were engineered. J-GFP-F corresponded to a GFP flanked by two leucine zippers (Jun and Fos); J/F-GFP was formed coexpressing a GFP fused to Jun leucine zipper (J-GFP) and a GFP fused to a Fos leucine zipper (F-GFP); and, finally, GFP was used as a control without any tag. All of them were expressed in Escherichia coli and formed IBs, where the aggregation tendency was especially high for J/F-GFP. Moreover, those IBs formed by J-GFP-F and J/F-GFP constructs were smaller, rougher, and more amorphous than GFP ones, increasing surface/mass ratio and, therefore, surface for protein release. Although the lipid and carbohydrate content were not reduced with the addition of leucine zippers, interesting differences were observed in the protein specific activity and conformation with the addition of Jun and Fos. Moreover, J-GFP-F and J/F-GFP nanoparticles were purer than GFP IBs in terms of protein content. This study proved that the use of leucine zippers strategy allows the formation of IBs with an increased aggregation ratio and protein purity, as we observed with the J/F-GFP approach, and the formation of IBs with a higher specific activity, in the case of J-GFP-F IBs. Thus, overall, the use of leucine zippers seems to be a good system for the production of IBs with more promising characteristics useful for pharma or biotech applications.

中文翻译：

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探索使用亮氨酸拉链来生成用于制药和生物技术应用的新型包涵体。


包涵体（IB）是具有生物活性的蛋白质聚集体，形成具有高稳定性和缓释行为的天然纳米颗粒。由于其性质，IB 已被探索用作组织工程中的生物催化剂，以及用于人类和动物治疗。为了提高这种纳米材料的生产和生物效率，对多种聚集标签进行了评估。然而，到目前为止，IBs 中与重组产物共存的细菌杂质（例如脂质和其他蛋白质）的研究还很少。这些杂质可能会严重限制 IB 应用的潜力，因为这是控制这些细菌纳米粒子的成分所必需的。因此，我们探索了使用亮氨酸拉链作为替代标签，不仅可以促进聚集，还可以促进具有改善的物理化学性质的新型 IB 样蛋白质纳米颗粒的生成。设计了三种不同的蛋白质构建体，命名为 GFP、J-GFP-F 和 J/F-GFP。 J-GFP-F 对应于侧翼有两个亮氨酸拉链（Jun 和 Fos）的 GFP； J/F-GFP形成为共表达与Jun亮氨酸拉链融合的GFP(J-GFP)和与Fos亮氨酸拉链融合的GFP(F-GFP)；最后，将 GFP 用作不带任何标签的对照。它们均在大肠杆菌中表达并形成IB，其中J/F-GFP的聚集趋势尤其高。此外，由 J-GFP-F 和 J/F-GFP 构建体形成的 IB 比 GFP 更小、更粗糙、更无定形，增加了表面/质量比，从而增加了蛋白质释放的表面。 尽管添加亮氨酸拉链并未降低脂质和碳水化合物含量，但添加 Jun 和 Fos 后，在蛋白质比活性和构象方面观察到有趣的差异。此外，J-GFP-F和J/F-GFP纳米粒子在蛋白质含量方面比GFP IB更纯。这项研究证明，正如我们用 J/F-GFP 方法观察到的那样，使用亮氨酸拉链策略可以形成具有更高聚集率和蛋白质纯度的 IB，并且可以形成具有更高比活性的 IB。 J-GFP-F IB。因此，总体而言，使用亮氨酸拉链似乎是生产 IB 的良好系统，其具有更有前景的特性，可用于制药或生物技术应用。