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Studies on the functions of chaperones participating cell morphology and on their relation with cell division

分子伴侣参与细胞形态的功能及其与细胞分裂关系的研究

基本信息

批准号：
10660097
负责人：
WATANABE Kunihiko
金额：
$ 2.18万
依托单位：
Kyoto Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660097/
关键词：
chaperone DanK cell division FtsZ HrcA HrcA フィラメント化

项目摘要

Overexpression and purification of Bacillus thermoglucosidasius KP1006 DnaK. Overexpression of the DnaK protein was proformed using Escherichia coli cells as a host. Purified DanK proteins consisted of monomer and two to four identical oligomers. This was different fromt the observation for E.coli DanK protein that has a dimeric structure. ATPase activity of B.thermoglucosidasius DnaK gradually decreased as the purity increased. This suggested the activity might correlate with DanJ and GrpE proteins.Analyses of proteins that have an interaction with B.thermoglucosidasius DanK. The interaction of B.thermoglucosidasius DanK and E.coli FtsZ was investigated using a two-hybird system. The interaction between the two molecules was successfully detected. Furthermore, the functional region related to the interaction is being determined by constructing various chimeric plasmids of two molecules on the two-hybird vectors.B.thermoglucosidasius HrcA, a negative control repressor for chaperone expression. HrcA proteins can function by binding to the CIRCE (controlling inverted repeat of chaperon expression) element. However, this protein tends to aggregate under the non-denaturing conditions. So we attempted to solubilize B.thermoglucosidasius HrcA and eventually succeeded in it with DNA. Moreover, we found the complex of B.thermoglucosidasius HrcA and DNA is stable at a lower range of temperature (below 50℃) and denatured at elevated temperature. Therefore, we suggested that B.thermoglucosidasius HrcA should function as a thermosensor to initiate the gene expression of the groEL and danK operons.

热葡糖苷酸芽孢杆菌KP1006 DnaK的过表达和纯化使用大肠杆菌细胞作为宿主进行DnaK蛋白的过表达。纯化的DanK蛋白由单体和两到四个相同的寡聚体组成。这与对具有二聚体结构的大肠杆菌DanK蛋白的观察不同。热葡萄糖苷酶B.thermoglucosidasius DnaK的ATP酶活性随着纯度的增加而逐渐降低。与热葡糖苷酶B.热葡糖苷酶DanK相互作用的蛋白质分析。利用双杂交系统研究了热葡糖苷酶B.thermoglucosidasius DanK和大肠杆菌FtsZ的相互作用。成功地检测到了两个分子之间的相互作用。此外，通过在双杂交载体上构建两种分子的各种嵌合质粒，确定了与相互作用相关的功能区域。B.thermoglucosidasius HrcA，伴侣表达的阴性对照阻遏物。HrcA蛋白可以通过结合CIRCE（控制伴侣表达的反向重复序列）元件来发挥功能。然而，这种蛋白质在非变性条件下倾向于聚集。因此，我们尝试溶解热葡糖苷酶B. hrcA，并最终用DNA成功地将其溶解。此外，我们还发现热葡糖苷酶B菌HrcA与DNA的复合物在较低温度范围内（低于50℃）是稳定的，在高温下会发生变性。因此，我们认为热葡萄糖苷酶B.thermoglucosidasius HrcA应该作为一种温度传感器来启动groEL和danK操纵子的基因表达。