Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, United States.
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, United States.
Methods Enzymol. 2023;689:3-38. doi: 10.1016/bs.mie.2023.05.010. Epub 2023 Jun 12.
Six cytochrome P450 enzymes are involved in human steroidogenesis, converting cholesterol to sex steroids, mineralocorticoids, and glucocorticoids. While early work was accomplished with steroidogenic P450 orthologs from more accessible sources, knowledge of basic biochemistry through successful drug design have been greatly facilitated by recombinantly-expressed, highly purified human versions of these membrane proteins. Many membrane proteins are difficult to express and purify and are unstable. Membrane P450 expression in E. coli has been facilitated by modification and/or truncation of the membrane-interacting N-terminus, while metal-affinity resins and histidine-tagging greatly facilitates purification. However, substantial optimization is still frequently required to maintain protein stability. Over time, a generalized three-column purification scheme has been developed and tweaked to generate substantial quantities of fully active, highly purified human cytochrome P450 enzymes that have made possible the application of many structural, biochemical, and biophysical techniques to elucidate the mysteries of these critical human enzymes.
六种细胞色素 P450 酶参与人体甾体激素生成，将胆固醇转化为性激素、盐皮质激素和糖皮质激素。虽然早期的工作是使用更易获得的甾体激素生成 P450 同源物完成的，但通过重组表达、高度纯化的这些膜蛋白的人类版本，成功的药物设计大大促进了基本生物化学知识的发展。许多膜蛋白难以表达和纯化，而且不稳定。通过修饰和/或截断与膜相互作用的 N 端，使大肠杆菌中的膜 P450 表达变得更加容易，而金属亲和树脂和组氨酸标记则极大地促进了纯化。然而，为了保持蛋白质稳定性，通常仍需要进行大量优化。随着时间的推移，已经开发并调整了一种通用的三柱纯化方案，以生成大量完全活性、高度纯化的人类细胞色素 P450 酶，这些酶使得应用许多结构、生化和生物物理技术来阐明这些关键人类酶的奥秘成为可能。
