Wolfgramm Hannes, Saade Christopher, Harms Marco, Busch Larissa M, Lange Josephine, Schedlowski Maximilian, Surmann Kristin, Gesell Salazar Manuela, Hentschker Christian, Steil Leif, Michalik Stephan, Völker Uwe, Reder Alexander
Interfaculty Institute of Genetics and Functional Genomics, Department of Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
Institute of Immunology, University Medicine Greifswald, Greifswald, Germany.
Microb Cell Fact. 2025 May 20;24(1):115. doi: 10.1186/s12934-025-02736-7.
Recombinant proteins facilitate and contribute to detailed studies of the virulence mechanisms and pathophysiology of the major human pathogen Staphylococcus aureus. Of particular interest are secreted virulence factors. However, due to their potential toxicity and specific post-translational processing, virulence factors are difficult targets for heterologous protein production. Purified proteins with native conformation and adequate purity can therefore often only be achieved by elaborate multi-step purification workflows. While homologous expression in S. aureus theoretically offers a promising alternative in this regard, its application remains limited due to the lack of systems that ensure both tightly controlled expression and subsequent efficient purification.
To bridge this gap, we present pTripleTREP as a versatile expression vector for S. aureus, which enables the homologous expression and purification of staphylococcal virulence factors. It features a strong SigA-dependent staphylococcal promoter overlapped by three tetracycline responsive elements (TRE), which ensures tight repression under control conditions and high expression levels upon induction of the target gene. This allowed very precise controlled production of the exemplary targets, serine protease-like protein A (SplA) and B (SplB). A simple single-step protein purification workflow using a Twin-Strep-tag and Strep-TactinXT coated magnetic beads yielded endotoxin-free Spl samples with purities above 99%. Thereby, the homologous production host facilitates native secretion and maturation without the need to engineer the target gene sequence. Proper signal peptide cleavage and the corresponding enzymatic activity of the generated protein products were confirmed for SplA and B.
The expression vector pTripleTREP adds an important element to the staphylococcal molecular toolbox, facilitating the tightly controlled homologous expression and rapid native purification of secreted staphylococcal virulence factors. The optimised architecture and genetic features of the vector additionally provide a solid background for further applications such as plasmid-based complementation or interaction studies. Thus, pTripleTREP will support research on the role of staphylococcal virulence factors, paving the way for future therapeutic strategies to combat this pathogen.
重组蛋白有助于深入研究主要人类病原体金黄色葡萄球菌的毒力机制和病理生理学。特别令人感兴趣的是分泌型毒力因子。然而，由于其潜在毒性和特定的翻译后加工过程，毒力因子是异源蛋白生产的困难靶点。因此，通常只有通过精心设计的多步纯化流程才能获得具有天然构象和足够纯度的纯化蛋白。虽然在金黄色葡萄球菌中的同源表达理论上在这方面提供了一个有前景的替代方法，但由于缺乏既能确保严格控制表达又能随后有效纯化的系统，其应用仍然有限。
为了弥补这一差距，我们提出了pTripleTREP作为一种用于金黄色葡萄球菌的通用表达载体，它能够同源表达和纯化葡萄球菌毒力因子。它具有一个由三个四环素反应元件（TRE）重叠的强大的依赖SigA的葡萄球菌启动子，这确保了在对照条件下的严格抑制以及在诱导靶基因时的高表达水平。这使得能够非常精确地控制生产示例性靶点，丝氨酸蛋白酶样蛋白A（SplA）和B（SplB）。使用双Strep标签和Strep-TactinXT包被的磁珠进行简单的单步蛋白纯化流程，得到了纯度高于99%且无内毒素的Spl样品。因此，同源生产宿主有助于天然分泌和成熟，而无需对靶基因序列进行工程改造。证实了SplA和B的信号肽正确切割以及所产生蛋白产物的相应酶活性。
表达载体pTripleTREP为葡萄球菌分子工具箱增添了一个重要元素，有助于严格控制同源表达并快速天然纯化分泌型葡萄球菌毒力因子。该载体优化的结构和遗传特征还为进一步的应用，如基于质粒的互补或相互作用研究，提供了坚实的基础。因此，pTripleTREP将支持对葡萄球菌毒力因子作用的研究，为未来对抗这种病原体的治疗策略铺平道路。
