Rancourt S L, Walker V K
Department of Biology, Queen's University, Kingston Canada.
Biochim Biophys Acta. 1990 Jul 6;1039(3):261-8. doi: 10.1016/0167-4838(90)90258-h.
Dihydrofolate reductase (DHFR) has been purified over 30,000-fold from Drosophila adults with a yield of 35%, using a combination of low pH extraction, (NH4)2SO4 precipitation, Sephadex gel filtration, Affi-Gel blue affinity chromatography, ion exchange and gel filtration FPLC. The Drosophila enzyme is a soluble, 17-22 kDa monomeric protein displaying the two pH optima characteristic of eukaryotic DHFRs. The sequence of the first 23 amino acids from the amino-terminal end of the protein shows that Drosophila DHFR is more homologous to the mosquito and vertebrate DHFRs than to the prokaryotic enzymes. However, the percent similarity between the two insect enzymes is not as close as expected when compared to the virtually identical initial sequence conservation of mammalian DHFRs.
利用低pH值提取、硫酸铵沉淀、葡聚糖凝胶过滤、Affi-Gel蓝亲和色谱、离子交换和凝胶过滤快速蛋白质液相色谱等方法相结合，已从果蝇成虫中纯化出二氢叶酸还原酶（DHFR），纯化倍数超过30000倍，产率为35%。果蝇酶是一种可溶性的17 - 22 kDa单体蛋白，具有真核DHFR的两个最适pH值特征。该蛋白质氨基末端的前23个氨基酸序列表明，果蝇DHFR与蚊子和脊椎动物的DHFR比与原核酶更同源。然而，与哺乳动物DHFR几乎相同的初始序列保守性相比，两种昆虫酶之间的相似百分比并不像预期的那么接近。
