Iida T, Iwabuchi T, Ideno A, Suzuki S, Maruyama T
Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, 026-0001, Iwate, Japan.
Gene. 2000 Oct 3;256(1-2):319-26. doi: 10.1016/s0378-1119(00)00378-4.
The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase). Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H. cutirubrum has an FKBP-type PPIase or not. In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H. cutirubrum and then sequenced. This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3kDa. Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs. Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp. KS-1. Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP. This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion. The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506. This FKBP suppressed aggregation of the unfolded protein.
嗜盐古菌红皮盐杆菌已被证明具有一种亲环蛋白型肽基脯氨酰顺反异构酶（PPIase）。由于大多数已研究的古菌基因组仅具有作为PPIase的FK506结合蛋白（FKBP）基因，因此尚不清楚红皮盐杆菌是否具有FKBP型PPIase。在本研究中，从红皮盐杆菌的基因组DNA中克隆了一个编码FKBP型PPIase的基因，然后进行了测序。该FKBP推测由303个氨基酸残基组成，分子量为33.3 kDa。将其氨基酸序列与其他已报道的FKBP的序列进行比对，结果表明它在对应于人类FKBP12的凸起和侧翼区域含有两个插入序列，这是古菌FKBP所共有的。其C端氨基酸序列比嗜热自养甲烷球菌和嗜热栖热菌KS-1的FKBP大约长130个氨基酸。在形成FK506结合口袋的14个保守氨基酸残基中，在该FKBP中仅发现了3个。该基因在大肠杆菌中作为与谷胱甘肽S-转移酶（GST）的融合蛋白表达，并且通过蛋白酶消化去除N端的GST部分。纯化的重组FKBP显示出较弱的PPIase活性，对FK506的敏感性较低。该FKBP抑制了未折叠蛋白的聚集。
