LaPorte D C, Chung T
J Biol Chem. 1985 Dec 5;260(28):15291-7.
The gene which codes for isocitrate dehydrogenase kinase/phosphatase of Escherichia coli, aceK, has been cloned. Physical and functional mapping of this clone indicated that both the isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase activities are encoded by an 1800-base pair sequence. This sequence produced a polypeptide with an apparent molecular weight of 66,000, which is identical to that of the purified protein. Since a protein of this size would require an 1800-base pair coding sequence, we conclude that isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase are expressed from a single gene. This strongly suggests that both activities reside on the same polypeptide chain. The cloning of aceK was made possible by the fortuitous addition of a second origin of replication to the expression vectors which were employed. These expression vectors were found to inhibit the growth of E. coli on the minimal acetate selective medium. The inclusion of a second origin of replication reduced the copy number and so reduced the inhibitory effects of these vectors. Control of the copy number through the addition of replication origins may have a general facility when manipulating plasmids which are potentially toxic to E. coli.
编码大肠杆菌异柠檬酸脱氢酶激酶/磷酸酶的基因aceK已被克隆。对该克隆的物理和功能图谱分析表明，异柠檬酸脱氢酶激酶和异柠檬酸脱氢酶磷酸酶活性均由一个1800碱基对的序列编码。该序列产生了一种表观分子量为66000的多肽，与纯化蛋白的分子量相同。由于这种大小的蛋白质需要一个1800碱基对的编码序列，我们得出结论，异柠檬酸脱氢酶激酶和异柠檬酸脱氢酶磷酸酶由单个基因表达。这强烈表明这两种活性存在于同一条多肽链上。aceK的克隆是由于在所用的表达载体中偶然添加了第二个复制起点而得以实现的。这些表达载体被发现会抑制大肠杆菌在最低限度乙酸盐选择培养基上的生长。添加第二个复制起点降低了拷贝数，从而降低了这些载体的抑制作用。在操作对大肠杆菌有潜在毒性的质粒时，通过添加复制起点来控制拷贝数可能具有普遍适用性。
