Recombinant protein expression in bacteria often leads to the formation of intracellular insoluble protein deposits, a major bottleneck for the production of soluble and active products. However, in recent years, these bacterial protein aggregates, commonly known as inclusion bodies (IBs), have been shown to be a source of stable and active protein for biotechnological and biomedical applications. The formation of these functional IBs is usually facilitated by the fusion of aggregation-prone peptides or proteins to the protein of interest, leading to the formation of amyloid-like nanostructures, where the functional protein is embedded. In order to offer an alternative to the classical amyloid-like IBs, here we develop functional IBs exploiting the coiled-coil fold. An in silico analysis of coiled-coil and aggregation propensities, net charge, and hydropathicity of different potential tags identified the natural homo-dimeric and anti-parallel coiled-coil ZapB bacterial protein as an optimal candidate to form assemblies in which the native state of the fused protein is preserved. The protein itself forms supramolecular fibrillar networks exhibiting only α-helix secondary structure. This non-amyloid self-assembly propensity allows generating innocuous IBs in which the recombinant protein of interest remains folded and functional, as demonstrated using two different fluorescent proteins. Here, we present a proof of concept for the use of a natural coiled-coil domain as a versatile tool for the production of functional IBs in bacteria. This α-helix-based strategy excludes any potential toxicity drawback that might arise from the amyloid nature of β-sheet-based IBs and renders highly active and homogeneous submicrometric particles.

中文翻译：

---

受螺旋线圈启发的功能性包涵体。

细菌中的重组蛋白表达常常导致细胞内不溶性蛋白沉积物的形成，这是生产可溶性活性产品的主要瓶颈。然而，近年来，这些细菌蛋白聚集体（通常称为包涵体（IB））已被证明是生物技术和生物医学应用的稳定和活性蛋白的来源。这些功能性 IB 的形成通常是通过将易于聚集的肽或蛋白质与目标蛋白质融合来促进的，从而形成淀粉样蛋白样纳米结构，其中嵌入功能性蛋白质。为了提供经典淀粉样蛋白 IB 的替代方案，我们利用卷曲螺旋折叠开发功能性 IB。对卷曲螺旋和聚集倾向、净电荷以及不同潜在标签的亲水性进行的计算机分析确定了天然同源二聚体和反平行卷曲螺旋 ZapB 细菌蛋白作为形成组装体的最佳候选者，其中天然状态的组装体融合蛋白被保留。该蛋白质本身形成超分子纤维网络，仅表现出 α 螺旋二级结构。这种非淀粉样蛋白自组装倾向可以产生无害的 IB，其中感兴趣的重组蛋白保持折叠和功能，如使用两种不同的荧光蛋白所证明的。在这里，我们提出了使用天然卷曲螺旋结构域作为在细菌中生产功能性 IB 的通用工具的概念证明。这种基于 α 螺旋的策略排除了基于 β 片层的 IB 的淀粉样蛋白性质可能引起的任何潜在毒性缺陷，并提供了高活性和均质的亚微米颗粒。