Jiang Shan, Çelen Gülce, Glatter Timo, Niederholtmeyer Henrike, Yuan Jing
Max Planck Institute for Terrestrial Microbiology and Center for Synthetic Microbiology, Marburg, Germany.
Max Planck Institute for Terrestrial Microbiology and Center for Synthetic Microbiology, Marburg, Germany; Technical University of Munich, Campus Straubing for Biotechnology and Sustainability, Straubing, Germany.
J Biol Chem. 2024 Nov;300(11):107850. doi: 10.1016/j.jbc.2024.107850. Epub 2024 Oct 1.
Numerous small proteins have been discovered across all domains of life, among which many are hydrophobic and predicted to localize to the cell membrane. Based on a few that are well-studied, small membrane proteins are regulators involved in various biological processes, such as cell signaling, nutrient transport, drug resistance, and stress response. However, the function of most identified small membrane proteins remains elusive. Their small size and hydrophobicity make protein production challenging, hindering function discovery. Here, we combined a cell-free system with lipid sponge droplets and synthesized small membrane proteins in vitro. Lipid sponge droplets contain a dense network of lipid bilayers, which accommodates and extracts newly synthesized small membrane proteins from the aqueous surroundings. Using small bacterial membrane proteins MgrB, SafA, and AcrZ as proof of principle, we showed that the in vitro-produced membrane proteins were functionally active, for example, modulating the activity of their target kinase as expected. The cell-free system produced small membrane proteins, including one from human, up to micromolar concentrations, indicating its high level of versatility and productivity. Furthermore, AcrZ produced in this system was used successfully for in vitro co-immunoprecipitations to identify interaction partners. This work presents a robust alternative approach for producing small membrane proteins, which opens a door to their function discovery in different domains of life.
在生命的所有领域都发现了许多小蛋白，其中许多是疏水的，预计定位于细胞膜。基于少数几个经过充分研究的小蛋白，小膜蛋白是参与各种生物过程的调节因子，如细胞信号传导、营养物质运输、耐药性和应激反应。然而，大多数已鉴定的小膜蛋白的功能仍然难以捉摸。它们的小尺寸和疏水性使得蛋白质生产具有挑战性，阻碍了功能的发现。在这里，我们将无细胞系统与脂质海绵微滴相结合，在体外合成了小膜蛋白。脂质海绵微滴包含密集的脂质双层网络，可容纳并从水性环境中提取新合成的小膜蛋白。以小细菌膜蛋白MgrB、SafA和AcrZ作为原理验证，我们表明体外产生的膜蛋白具有功能活性，例如，如预期那样调节其靶激酶的活性。该无细胞系统产生的小膜蛋白，包括一种来自人类的小膜蛋白，浓度高达微摩尔级，表明其具有高度的通用性和生产力。此外，在该系统中产生的AcrZ成功用于体外免疫共沉淀以鉴定相互作用伙伴。这项工作提出了一种生产小膜蛋白的强大替代方法，为在生命的不同领域发现其功能打开了一扇门。
