棉铃虫中肠微粒体P450的分离纯化

Partial purification of microsomal P450s from midgut of the cotton bollworm, Helicoverpa armigera

为深入研究棉铃虫Helicoverpaarmigera细胞色素P450的结构与功能,需要分离不同型的P450蛋白。作者建立了适用于棉铃虫中肠微粒体P450的纯化方法,包括聚乙二醇8000(PEG8000)沉淀、高效疏水作用色谱(HPHIC)和高效离子交换色谱(HPIEC)等连续分离步骤。SDS_PAGE(银染)显示,棉铃虫中肠微粒体经以上步骤分离纯化后,在含P450的馏分中检测出分子量分别为58kD、47kD、56kD和45kD的4条蛋白带。P450的回收率为14.3%,比含量提高了39倍。

A protocol comprising PEG8000 precipitation, high performance hydrophobic interaction chromatography (HPHIC) and high performance ion exchange chromatography (HPIEC) for separation and purification of cytochrome P450 in the cotton bollworm, Helicoverpa armigera, was introduced. PEG8000 precipitation was found suitable for pre-fractionation of the midgut microsomes P450 in H. armigera. The favorable conditions of HPHIC and HPIEC for purifying microsomal P450 were established. Following purification, four proteins with molecular weight of 58 kD, 47 kD, 56 kD and 45 kD were detected in the fractions with cytochrome P450 absorbance peak by silver-staining SDS-PAGE gel. The recovery rate of P450s was 14.3%, and the P450 purity was increased by 39-fold.