Production of human mitochondrial ABC transporters for structural and biochemical studies

人类线粒体内膜有三种ABC转运蛋白：ABCB 7参与 在铁转运中（突变导致X连锁铁粒幼细胞性贫血伴共济失调），ABCB 8参与 调节线粒体K+通道和保护免受阿霉素毒性，以及ABCB 10， 对红细胞发育、保护免受氧化应激是必需的， 导致小鼠胚胎死亡。尽管它们的重要性显而易见，但几乎没有进行过分子研究 这些人类线粒体转运蛋白，主要是因为人类膜蛋白的生产， 难我们相信，促进这些转运体的生产将加速所需的研究， 更好地了解其功能背后的分子机制。人类ABC转运蛋白 只有在昂贵和耗时的真核系统中才能产生，直到我们的团队最近才有了 在细菌中产生ABCB 10的想法。我们的初步研究表明，细菌中产生的ABCB 10是 不降低在这里，我们建议使用这种新的方法，简单，快速，廉价的生产 ABCB7和ABCB8。目的1将开发表达和纯化功能性ABCB 7的方法， 细菌中的ABCB8。目的2将优化重组纯化的转运蛋白到脂质中的方法 双层（纳米盘和脂质体）以进行功能测定。我们的建议是高风险的，因为我们将 测试在细菌中生产这些人类线粒体转运蛋白的可行性。这项建议是 重要的是，它将提供优化的生产和功能测试方法， 重要的人类蛋白质，几乎没有被研究，这将是高回报。我们的经验 ABC转运蛋白分子研究小组，结合这些新的方法，将提供一个 一个更雄心勃勃的R01建议，旨在研究的基本分子机制， 这些人类线粒体转运蛋白。

Project Summary The human mitochondrial inner membrane has three ABC transporters: ABCB7 implicated in iron transport (with mutations causing X-linked sideroblastic anemia with ataxia), ABCB8 involved in regulation of a mitochondrial K+ channel and protection against doxorubicin toxicity, and ABCB10, which is essential for red blood cell development, protection against oxidative stress and whose complete absence leads to death of mice embryos. Despite their clear importance, little molecular research has been done on these human mitochondrial transporters, mainly because production of human membrane proteins is difficult. We believe that facilitating the production of these transporters will accelerate the research needed for a better molecular understanding of the mechanisms underlying their function. Human ABC transporters had only been produced in expensive and time-consuming eukaryotic systems until our group recently had the idea of producing ABCB10 in bacteria. Our preliminary studies show that ABCB10 produced in bacteria is functional. Here, we propose to use this novel approach for the easy, fast, and inexpensive production of ABCB7 and ABCB8. Aim 1 will develop methodologies for expression and purification of functional ABCB7 and ABCB8 in bacteria. Aim 2 will optimize methodologies for reconstitution of the purified transporters into a lipid bilayer (nanodiscs and liposomes) to perform functional assays. Our proposal is of high risk because we will test the feasibility of producing these human mitochondrial transporters in bacteria. This proposal is significant because it will provide optimized methodologies for production and functional testing of two important human proteins that have barely been studied, which will be of high reward. The experience of our group on ABC transporters molecular research, combined with these new methodologies, will provide a pathway for a more ambitious R01 proposal aimed at studying the fundamental molecular mechanisms of these human mitochondrial transporters.