Institute of Microbiology, Heinrich-Heine University, Düsseldorf, Germany.
Center for Synthetic Microbiology, Philipps-University Marburg, Marburg, Germany.
Methods Mol Biol. 2025;2850:229-249. doi: 10.1007/978-1-0716-4220-7_13.
Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.
重组蛋白生产在分子生物学中至关重要，通过对纯化样品进行生物物理、生化和结构分析，能够深入了解细胞过程。大量生物分子的需求常常带来挑战，特别是对于真核蛋白。大肠杆菌表达系统已经发展到能够解决这些问题，提供了可溶性标签、翻译后修饰能力和模块化质粒文库等先进功能。然而，现有的工具往往很复杂，这限制了它们的可及性，并需要简化的系统来在标准化条件下进行快速筛选。基于 Golden Gate 克隆方法，我们开发了一种简单的“一步法”，使用策略性选择的蛋白纯化标签（如六组氨酸、SUMO、MBP、GST 和 GB1）生成表达构建体，以提高蛋白的可溶性和表达量。该系统通过 mScarlet 荧光实现可视化的候选物筛选，并且可以通过 TEV 蛋白酶切割去除可溶性标签。我们提供了一个全面的方案，包括寡核苷酸设计、克隆、表达、His 标签亲和层析和分子筛层析。因此，该方法简化了原核和真核蛋白的生产，使得具有基本蛋白生化设备的标准分子生物学实验室也能够使用。
