Piselli Claudio
Department of Protein Evolution, Departmental Research Group Molecular Recognition and Catalysis, Max Planck Institute for Biology, Tubingen, Germany.
Nat Protoc. 2025 Feb;20(2):462-479. doi: 10.1038/s41596-024-01055-2. Epub 2024 Oct 4.
The recombinant expression, isolation and characterization of pore-forming proteins is one of the most commonly used strategies for understanding the permeability properties of the biological membrane into which they are embedded. This protocol describes how to quantify the expression of your protein of interest and use this information to optimize its production using the Escherichia coli strain BL21Gold(de3)ΔABCF. It explains with a step-by-step approach how to separate the bacterial compartments according to their solubility and how to extract your protein of interest in its native conformation using detergent solutions. Finally, it describes how to improve its purity via ion-exchange chromatography and insert the purified porins into outer membrane vesicles, from which they can be copurified. The protocol is simpler and less empirical than those described for most channel-forming membrane proteins and also provides a solid foundation for the isolation of soluble proteins. Several parameters can be optimized on a case-by-case basis: expression time and temperature, concentration of the inducer, nature and concentration of the detergent, incubation time and temperature, pH and ionic strength of the purification buffers. This protocol is effective with prokaryotic channel-forming membrane proteins and can be employed for the production of pore-forming proteins from chloroplasts, mitochondria or eukaryotes in general. With minor optimization, this protocol can be adapted for the isolation of receptors, carrier, pumps or any other membrane-active proteins.
孔形成蛋白的重组表达、分离和表征是理解其嵌入的生物膜通透性特性最常用的策略之一。本方案描述了如何定量目标蛋白的表达，并利用这些信息使用大肠杆菌BL21Gold(de3)ΔABCF菌株优化其生产。它逐步解释了如何根据细菌各部分的溶解性进行分离，以及如何使用去污剂溶液以天然构象提取目标蛋白。最后，它描述了如何通过离子交换色谱提高其纯度，并将纯化的孔蛋白插入外膜囊泡中，从而实现共纯化。该方案比大多数形成通道的膜蛋白所描述的方案更简单、更少依赖经验，也为可溶性蛋白的分离提供了坚实的基础。几个参数可以根据具体情况进行优化：表达时间和温度、诱导剂浓度、去污剂的性质和浓度、孵育时间和温度、纯化缓冲液的pH值和离子强度。该方案对原核生物形成通道的膜蛋白有效，一般可用于生产叶绿体、线粒体或真核生物的孔形成蛋白。经过微小的优化，该方案可适用于受体、载体、泵或任何其他膜活性蛋白的分离。
