Department of Ruminant Production, Institute of Agriculture and Food Research and Technology (IRTA), 08140, Caldes de Montbui, Spain.
Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via L. Giorgieri 1, 34127, Trieste, Italy.
Microb Cell Fact. 2020 Sep 4;19(1):175. doi: 10.1186/s12934-020-01425-x.
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.
包涵体（IBs）是具有生物活性的蛋白质聚集体，形成具有高稳定性和缓慢释放特性的天然纳米颗粒。由于其性质，IBs 已被探索用于作为生物催化剂、组织工程以及人类和动物治疗。为了提高这种纳米材料的生产和生物学效率，已经评估了广泛的聚集标签。然而，到目前为止，IB 中存在的细菌杂质（如脂质和其他与重组产物共存的蛋白质）研究得还很少。这些杂质可能会严重限制 IB 应用的潜力，因此有必要控制这些细菌纳米颗粒的组成。因此，我们探索了使用亮氨酸拉链作为替代标签，不仅促进聚集，而且还生成具有改进的物理化学性质的新型 IB 样蛋白纳米颗粒。
设计了三种不同的蛋白质构建体，分别命名为 GFP、J-GFP-F 和 J/F-GFP。J-GFP-F 对应于 GFP 两侧侧翼的两个亮氨酸拉链（Jun 和 Fos）；J/F-GFP 是由 GFP 融合到 Jun 亮氨酸拉链（J-GFP）和 GFP 融合到 Fos 亮氨酸拉链（F-GFP）形成的共表达；最后 GFP 作为没有任何标签的对照。它们都在大肠杆菌中表达并形成 IBs，其中 J/F-GFP 的聚集趋势特别高。此外，与 GFP 相比，由 J-GFP-F 和 J/F-GFP 构建体形成的 IBs 更小、更粗糙且更无定形，增加了表面/质量比，从而增加了蛋白质释放的表面积。尽管添加亮氨酸拉链并没有降低脂质和碳水化合物的含量，但在添加 Jun 和 Fos 时，观察到蛋白质比活度和构象有有趣的差异。此外，就蛋白质含量而言，J-GFP-F 和 J/F-GFP 纳米颗粒比 GFP IBs 更纯。
这项研究证明，使用亮氨酸拉链策略可以形成具有更高聚集比和蛋白质纯度的 IBs，如我们在 J/F-GFP 方法中观察到的那样，并且可以形成具有更高比活度的 IBs，如 J-GFP-F IBs 中观察到的那样。因此，总体而言，使用亮氨酸拉链似乎是一种生产具有更有前途的特性的 IBs 的良好系统，这些特性可用于制药或生物技术应用。
