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

热葡萄糖苷芽孢杆菌KP1006DNAK的高效表达及纯化以大肠杆菌细胞为宿主进行了DNAK蛋白的高效表达。纯化的Dank蛋白由单体和2~4个相同的寡聚体组成。这与观察到的具有二聚体结构的E.coliDank蛋白不同。热葡萄糖苷芽孢杆菌DNAK的ATPase活性随纯度的增加而逐渐降低。这表明该活性可能与DanJ和GRPE蛋白有关。对与热葡萄糖苷杆菌相互作用的蛋白质的分析。用双杂交系统研究了嗜热葡萄糖苷杆菌Dank与大肠杆菌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，与相互作用相关的功能区是通过在双杂交载体上构建不同的两个分子的嵌合质粒来确定的。HrcA蛋白可以通过与CERCE(控制伴侣反向重复表达)元件结合来发挥功能。然而，这种蛋白质往往在非变性条件下聚集。因此，我们尝试溶解热葡萄糖苷杆菌HrcA，并最终用DNA将其溶解成功。此外，我们还发现，热葡萄糖苷杆菌HrcA与DNA的复合物在较低的温度范围内(低于50℃)是稳定的，在较高的温度下会发生变性。因此，我们建议热葡萄糖苷杆菌HrcA可以作为一个温度传感器来启动GroEL和Dank操纵子的基因表达。

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.