Faculty of Agriculture, Kagoshima University, Kagoshima, Japan.
Int J Biol Macromol. 2011 Nov 1;49(4):778-83. doi: 10.1016/j.ijbiomac.2011.07.014. Epub 2011 Aug 5.
We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.
我们之前曾报道过，含有六聚组氨酸的氨基末端延伸序列有助于从极端嗜盐古菌盐杆菌（NDK）中六聚核苷酸二磷酸激酶的复性和组装。在这项研究中，我们在标签序列和 NDK 分子内部都进行了各种突变。用六聚丝氨酸取代六聚组氨酸的 SerNDK 没有表现出折叠促进作用。此外，NDK 中的 Asp58 和 Asp63 均被 Gly 取代的 HisD58GD63G 也没有表现出复性增强。这些结果表明 His 标签中的六聚组氨酸与 Asp58 或 Asp63 或两者相互作用以促进折叠。此外，在低盐溶液中形成二聚体的 G114D 突变体，通过 His 标签强烈稳定，形成稳定的六聚体。
