Sueyoshi T, Park L J, Moore R, Juvonen R O, Negishi M
Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Arch Biochem Biophys. 1995 Sep 10;322(1):265-71. doi: 10.1006/abbi.1995.1461.
Peptitergented P450 2a-4 (Pepti-P450), a water-soluble form of the mouse microsomal P450 2a-4, was genetically engineered and expressed in Escherichia coli. The NH2-terminal hydrophobic sequence (positions 2 to 19) of Pepti-P450 was replaced by a peptitergent PD1, amphipathic peptide consisting of 24 residues (C. E. Schafmeister, L. J. Miercke, and R. M. Stroud (1993) Science 262, 734-738). The expression level of Pepti-P450 (90,000 molecules/cell) was at least four times greater than that of wild-type P450 2a-4. Since Pepti-P450 was quite stable and was expressed as a peripheral membrane protein, it can be easily purified from the membrane fraction treated with Na2CO3 without using any detergents during the chromatographic steps. The purified Pepti-P450 retained the spectral and catalytic properties of the unmodified enzyme with a similar Km value for steroid 15 alpha-hydroxylase activity (19.7 microM in comparison to 14.2 microM of the wild-type). Gel permeation chromatography showed that the purified Pepti-P450 in the detergent-free buffer was an oligomer with an approximate molecular mass of 450 kDa. The replacement of the hydrophobic anchor domain with an amphipathic helix such as peptitergent, therefore, may provide a general method for engineering membrane-bound P450s to soluble enzymes.
肽洗涤剂化的P450 2a - 4（Pepti - P450）是小鼠微粒体P450 2a - 4的水溶性形式，通过基因工程构建并在大肠杆菌中表达。Pepti - P450的NH2末端疏水序列（第2至19位）被肽洗涤剂PD1取代，PD1是一种由24个残基组成的两亲性肽（C. E. Schafmeister、L. J. Miercke和R. M. Stroud（1993年）《科学》262卷，734 - 738页）。Pepti - P450的表达水平（90,000个分子/细胞）至少是野生型P450 2a - 4的四倍。由于Pepti - P450相当稳定且作为外周膜蛋白表达，在色谱步骤中无需使用任何洗涤剂，就能轻松从用碳酸钠处理过的膜部分中纯化出来。纯化后的Pepti - P450保留了未修饰酶的光谱和催化特性，对于类固醇15α - 羟化酶活性具有相似的Km值（19.7微摩尔，而野生型为14.2微摩尔）。凝胶渗透色谱显示，在无洗涤剂缓冲液中的纯化Pepti - P450是一种近似分子量为450 kDa的寡聚体。因此，用两亲性螺旋如肽洗涤剂取代疏水锚定结构域，可能为将膜结合型P450工程改造为可溶性酶提供一种通用方法。
