Bhattacharyya D, Tano K, Bunick G J, Uberbacher E C, Behnke W D, Mitra S
University of Tennessee Graduate School of Biomedical Sciences, Oak Ridge 37831.
Nucleic Acids Res. 1988 Jul 25;16(14A):6397-410. doi: 10.1093/nar/16.14.6397.
The E. coli Ada protein (O6-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E280 nm (1%)) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from O6-methylguanine in DNA. Its reaction with O6-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 x 10(9) M-1 min-1 at O degree C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low alpha-helical content and the radius of gyration of 23 A indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.
大肠杆菌Ada蛋白（O6-甲基鸟嘌呤-DNA甲基转移酶）已使用高表达载体进行纯化，每升大肠杆菌培养物的产量约为3毫克。这种39千道尔顿的蛋白质的消光系数（E280 nm（1%））为5.3。其等电点为7.1，低于根据氨基酸含量预测的值。均一的Ada蛋白作为DNA中O6-甲基鸟嘌呤的甲基受体具有完全活性。在0℃下，它与合成DNA中的O6-甲基鸟嘌呤反应的二级速率常数为1.1×10⁹ M⁻¹ min⁻¹。天然形式和在半胱氨酸-69处甲基化的蛋白均为单体。圆二色谱表明α-螺旋含量较低，回转半径为23埃表明其形状紧凑、呈球状。该蛋白的中间区域对多种蛋白酶敏感，包括大肠杆菌中的一种内源性活性蛋白酶，这表明该蛋白由通过铰链区连接的N端和C端结构域组成。大肠杆菌B中的这种蛋白酶水平高于K12。
