Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

膜蛋白在健康和疾病中发挥重要作用。尽管它们很重要，但膜蛋白的研究受到其成功表达、纯化和稳定化所固有的困难的极大限制，一旦它们从细胞膜中提取出来。此外，人膜蛋白的表达通常需要使用昂贵和/或耗时的真核系统，因此它们在细菌中的成功表达显然有利于实验研究。此外，由于脂质可以对膜蛋白的活性产生关键影响，并且考虑到线粒体内膜和细菌质膜之间的组成相似性，大肠杆菌中线粒体膜蛋白的产生代表一个合乎逻辑的选择。在这里，我们提出了一种在大肠杆菌中生产人类线粒体 ATP 结合盒 (ABC) 转运蛋白的新方案。三种已知的人类线粒体 ABC 转运蛋白的功能尚不完全清楚，但昆虫细胞中产生的 ABCB10 的 X 射线晶体学模型是可用的。我们已经成功地从大肠杆菌中表达和纯化了 ABCB10 。产量接近于我们实验室在类似条件下常规生产的另一种细菌 ABC 转运蛋白的产量。此外，我们可以有效地将洗涤剂纯化的 ABCB10 重组为脂质纳米圆盘。测量大肠杆菌中产生的 ABCB10 的 ATP 酶活性显示与其他人类 ABC 转运蛋白相似的 ATP 水解速率。这种新颖的协议有助于生产这种用于生化、结构和功能分析的人类线粒体转运蛋白，并且可能会针对其他线粒体转运蛋白的生产进行调整。