Ishii Y, Sonezaki S, Iwasaki Y, Tauchi E, Shingu Y, Okita K, Ogawa H I, Kato Y, Kondo A
Department of Applied Chemistry, Faculty of Engineering, Kyushu Institute of Technology, Sensuicho, Tobata, Kitakyushu, 804, Japan.
J Biochem. 1998 Oct;124(4):842-7. doi: 10.1093/oxfordjournals.jbchem.a022188.
DnaJ is a molecular chaperone, which contains a zinc finger-like motif and cooperates with DnaK to mediate the folding of newly synthesized and denatured proteins. DnaJ was overproduced and purified using the maltose binding protein (MBP) fusion vector. The fusion protein (MBP-DnaJ) was expressed in a soluble form in Escherichia coli and purified to homogeneity using amylose resin in a single step. The UV-visible absorption spectrum of MBP-DnaJ showed peaks at 355 and 475 nm. Moreover, these absorption peaks disappeared upon treatment with ethylenediaminetetraacetic acid (EDTA) or p-hydroxymercuriphenylsulfonic acid (PMPS). Inductively coupled plasma (ICP) spectrometry demonstrated that MBP-DnaJ contains Fe ions as well as Zn ions. MBP-DnaJ mediated the replication of the lambda phage in vivo, stimulated the ATPase activity of DnaK and prevented the aggregation of denatured rhodanase, indicating that fusion of MBP to the N-terminal of DnaJ does not affect the functions of DnaJ. To study the roles of bound metal ions, metal-free MBP-DnaJ, and MBP-DnaJ containing 2 Zn ions were prepared. MBP-DnaJ containing Fe and Zn ions, and MBP-DnaJ containing 2 Zn ions stimulated the ATPase activity of DnaK, prevented the aggregation of denatured rhodanase and bound to DNA to similar extents. On the other hand, metal-free MBP-DnaJ showed much lower DNA-binding ability and lower ability to prevent rhodanese aggregation. Therefore, the bound metal species do not affect the function of the zinc finger-like motif of DnaJ, whereas removal of the metal ions from DnaJ diminishes its binding ability as to DNA and denatured proteins.
DnaJ是一种分子伴侣，它含有一个锌指样基序，并与DnaK协同作用以介导新合成蛋白质和变性蛋白质的折叠。使用麦芽糖结合蛋白（MBP）融合载体过量表达并纯化了DnaJ。融合蛋白（MBP-DnaJ）在大肠杆菌中以可溶形式表达，并使用直链淀粉树脂一步纯化至均一性。MBP-DnaJ的紫外可见吸收光谱在355和475nm处有峰值。此外，用乙二胺四乙酸（EDTA）或对羟基汞苯磺酸（PMPS）处理后，这些吸收峰消失。电感耦合等离子体（ICP）光谱表明，MBP-DnaJ含有铁离子以及锌离子。MBP-DnaJ在体内介导了λ噬菌体的复制，刺激了DnaK的ATP酶活性，并防止了变性硫氰酸酶的聚集，表明MBP与DnaJ的N端融合不影响DnaJ的功能。为了研究结合的金属离子的作用，制备了无金属的MBP-DnaJ和含有2个锌离子的MBP-DnaJ。含有铁和锌离子的MBP-DnaJ以及含有2个锌离子的MBP-DnaJ在相似程度上刺激了DnaK的ATP酶活性，防止了变性硫氰酸酶的聚集并与DNA结合。另一方面，无金属的MBP-DnaJ显示出低得多的DNA结合能力和较低的防止硫氰酸酶聚集的能力。因此，结合的金属种类不影响DnaJ的锌指样基序的功能，而从DnaJ中去除金属离子会降低其与DNA和变性蛋白质的结合能力。
