Purification of Rts1 RepA protein and its characterization

1. RepA蛋白的纯化。将编码Rts 1复制所必需的RepA蛋白的repA基因插入到载体质粒pKK 223 -3中，从而在大肠杆菌中在tac启动子控制下表达repA。coli JM 103。用CM-Sephadex柱、磷酸纤维素柱和Affigel蓝柱层析纯化从10升经IPTC诱导的细胞培养物中获得的粗裂解物中的RepA。纯化的RepA氨基酸组成与根据repA序列推测的氨基酸组成一致. RepA与DNA的结合（通过DNase I保护试验检测）：纯化的RepA被证实与repA启动子的紧邻上游区域（这将是操纵子）以及incII和GATC盒之间的10-bp部分（除了incI和incII重复序列之外）强烈结合。10-bp区域可能是Rts 1复制的起始位点. ori（Rts 1）的分离。当RepA从mini-Rts 1质粒体内研究中反式提供时，跨越坐标1441至1194的mini-Rts 1亚区能够启动再定位。ori（Rts 1）中含有incII序列，沿着含有GATC和DnaA盒.缺失RepA的C末端。构建了一系列编码RepA衍生物的repA突变体，所述RepA衍生物含有取代羧基末端6个氨基酸的寡肽。这些修饰的RepA完全不能激活ori（Rts 1）。其中一个RepA衍生物对mini-Rts 1质粒具有弱但明显的不相容性，这可能是由于RepA与repA的操纵子结合所致。

1. Purification of RepA protein. The repA gene, encoding RepA protein that is essential for the replication of Rts1, was inserted into a vector plasmid pKK223-3, thereby expressing repA under the control of tac promoter in E. coli JM103. RepA in a crude lysate obtained from 10 liters culture of the cells induced by IPTC was purified through chromatographies using CM-sephadex, phosphocellulose and Affigel blue columns. The amino acid composition of the purified RepA was in agree with the composiion deduced from the repA sequence.2. Binding of RepA to DNA (examined by DNase I protection assay).The purified RepA was confirmed to bind strongly to the immediately upstream region of promoter of repA(which would be an operator) and a 10-bp portion between incII and GATC box besides incI and incII repeated sequences. The 10-bp region would be a possible site of origin of Rts1 replication.3. Isolation of ori(Rts1). A mini-Rts1 subregion that spans the coordinates 1441 to 1194 was capable of initiating relpication when RepA was supplied in trans from mini-Rts1 plasmid in vivo study. In the ori(Rts1), incII sequences along with GATC and DnaA boxes are contained.4. Deletion of C terminus of RepA. A series of repA mutants that encode RepA derivatives containing oligopeptide substitutions in place of the carboxyl terminal six amino acids were constructed. These modified RepA could not activate ori(Rts1) at all. One of the RepA derivatives, however, maintained a weak but evident incompatibility toward mini-Rts1 plasmid, which may be caused by binding of the RepA to operator of repA.