A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function
A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function
复制标题
DOI：
10.1006/abbi.2001.2574
复制
发表时间：
2001-11-01
影响因子：
3.9
通讯作者：
Halpert, JR
中科院分区：
文献类型：
--
作者：
Scott, EE;Spatzenegger, M;Halpert, JR
四种细胞色素 P450 2B 中的疏水性跨膜结构域被去除 (Delta3-21)，并在 N 末端取代了几个正电荷以增加表达和溶解度。将组氨酸残基附加到 C 末端以简化纯化。截短的蛋白质在大肠杆菌中高度表达，可以使用高盐条件从膜上释放，并使用单个 Ni2+-琼脂糖柱从该级分中纯化至高达 19 nmol P450/mg 蛋白质的特定含量。凝胶过滤显示，截短的 P450 2B1 在没有洗涤剂的情况下形成六聚体的单分散溶液，并且在 0.25% 胆酸钠中形成 > 95% 的单体。所有截短的蛋白质，包括人 2B6，都具有 7-乙氧基-4-三氟甲基香豆素的活性，并且截短的 2B1 显示保留了睾酮羟基化的天然区域特异性和立体特异性。这些数据表明，N 末端的修饰可产生高水平的正确折叠的 P450s 2B，且溶解度增加，适合功能和结构分析。 (C) 2001 年学术出版社。
The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni2+-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and > 95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stercospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis. (C) 2001 Academic Press.