Padas P M, Wilson K S, Vorgias C E
European Molecular Biology Laboratory, c/o DESY, Hamburg, Germany.
Gene. 1992 Aug 1;117(1):39-44. doi: 10.1016/0378-1119(92)90487-a.
The major histone-like bacterial protein (HU)-encoding genes (hup) from five different Bacilli have been cloned, sequenced and overexpressed in Escherichia coli. The five Bacilli selected are closely related, but have different optimum growth temperatures: greater than 70 degrees C for Bacillus caldolyticus and B. caldotenax; 60-65 degrees C for B. stearothermophilus (Bst); 37 degrees C for B. subtilis and 30 degrees C for B. globigii. The deduced amino acid (aa) sequences from the three thermophiles are identical. Those from the two mesophiles are also identical and differ from those of the thermophiles at eleven aa positions. The mesophilic proteins have an extra two aa at the C terminus. Cells harbouring plasmids containing the hup genes can produce HU. An efficient purification scheme using cation-exchange chromatography and fast protein liquid chromatography is presented. This gives approx. 30-40 mg of more than 95% pure Bst HU per litre of E. coli culture.
已从五种不同的芽孢杆菌中克隆、测序并在大肠杆菌中过表达了主要的类组蛋白细菌蛋白（HU）编码基因（hup）。所选的五种芽孢杆菌亲缘关系密切，但具有不同的最适生长温度：嗜热解芽孢杆菌和嗜热栖热芽孢杆菌的最适生长温度高于70℃；嗜热脂肪芽孢杆菌（Bst）为60 - 65℃；枯草芽孢杆菌为37℃，球形芽孢杆菌为30℃。三种嗜热菌的推导氨基酸（aa）序列相同。两种嗜温菌的序列也相同，并且在11个氨基酸位置上与嗜热菌的序列不同。嗜温蛋白在C末端多了两个氨基酸。携带含有hup基因质粒的细胞可以产生HU。本文介绍了一种使用阳离子交换色谱和快速蛋白质液相色谱的高效纯化方案。每升大肠杆菌培养物可得到约30 - 40毫克纯度超过95%的Bst HU。
