Papa Johanna E, Vaughn Lindsay R, Bartholomew-Schoch Jackson L, Stone Emma K, Culpepper Megen A, Reddish Michael J
Department of Chemistry and Fermentation Sciences, Appalachian State University, Boone, NC, USA.
Department of Chemistry and Fermentation Sciences, Appalachian State University, Boone, NC, USA.
Protein Expr Purif. 2025 Sep;233:106748. doi: 10.1016/j.pep.2025.106748. Epub 2025 May 26.
Human mitochondrial cytochrome P450 27A1 is a monooxygenase enzyme that oxidizes bile acids and other sterol derivatives. The enzyme plays an important role in sterol metabolism and is a potential target for clinical therapies related to metabolic conditions and certain cancers. To support the development of such therapies, detailed structural and functional studies of the enzyme should be pursued. Producing large quantities of purified, recombinant enzyme would enable these studies. Recombinant production of human cytochrome P450 27A1 in E. coli is challenging due to the enzyme being membrane associated. This work explores the optimization of human cytochrome P450 27A1 expression in E. coli by systematically testing the effects of cell strain, expression temperature, concentrations of induction reagents, and expression times. Western blot analysis is used to investigate the effects of variable changes prior to purification. E. coli cell strain (switching to C41(DE3)) appears to have the largest positive effect on overall yield. Increasing δ-aminolevulinic acid concentration (induces heme synthesis) also leads to significantly increased yields. Decreasing expression time decreases the amount of higher order cytochrome P450 aggregates that are formed. The combination of these changes is a more robust expression protocol with three major advantages: decreased expression time, lower aggregate to monomer ratios, and increased overall yield.
人类线粒体细胞色素P450 27A1是一种单加氧酶，可氧化胆汁酸和其他甾醇衍生物。该酶在甾醇代谢中起重要作用，是与代谢状况和某些癌症相关的临床治疗的潜在靶点。为了支持此类治疗方法的开发，应开展对该酶详细的结构和功能研究。大量生产纯化的重组酶将使这些研究成为可能。由于该酶与膜相关，在大肠杆菌中重组生产人细胞色素P450 27A1具有挑战性。这项工作通过系统测试细胞菌株、表达温度、诱导试剂浓度和表达时间的影响，探索了在大肠杆菌中优化人细胞色素P450 27A1表达的方法。在纯化之前，使用蛋白质免疫印迹分析来研究变量变化的影响。大肠杆菌细胞菌株（改用C41(DE3)）似乎对总产量有最大的正向影响。增加δ-氨基乙酰丙酸浓度（诱导血红素合成）也会导致产量显著提高。缩短表达时间可减少形成的高阶细胞色素P450聚集体的数量。这些变化的组合是一种更稳健的表达方案，具有三个主要优点：缩短表达时间、降低聚集体与单体的比例以及提高总产量。
