Tokunaga H, Hara S, Arakawa T, Ishibashi M, Gupta R S, Tokunaga M
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Japan.
J Protein Chem. 1999 Nov;18(8):837-44. doi: 10.1023/a:1020675128201.
The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH2-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS-PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS-PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.
在嗜盐古细菌红皮盐杆菌（Halobacterium cutirubrum）生长温度升高时，六种蛋白质的合成水平增加。其中一种蛋白质在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上的表观分子量为97 kDa，在4 M NaCl存在的情况下与ATP-琼脂糖柱结合，但在无盐情况下不结合，这表明该蛋白质仅在高盐浓度下保留其ATP结合活性。该蛋白质的NH2末端序列以及覆盖总残基数1/3的胰蛋白酶肽段的内部序列与从红皮盐杆菌分离的dnaK基因的核苷酸序列推导的序列一致。结果强烈表明，这种表观97 kDa的蛋白质是dnaK的基因产物，尽管根据核苷酸序列计算的分子量仅为68,495，远小于通过SDS-PAGE测定的该蛋白质的值。弗格森图分析表明，该蛋白质在SDS-PAGE上表现出异常迁移率。我们通过从ATP-琼脂糖柱上逐步洗脱，将DnaK同源物纯化至大于90%的纯度。
