Purification of Rts1 RepA protein and its characterization

1.REPA蛋白的纯化。将编码rts1复制所必需的repa蛋白的repa基因插入到载体pKK223-3中，在E.coliJM103中实现了tac启动子控制下的repa表达。通过CM-Sephadex、磷酸纤维素柱和Affigel蓝柱层析，从IPTC诱导的10升培养细胞中获得了粗裂液中的REPA。纯化的REPA的氨基酸组成与根据REPA序列推定的组成一致。纯化的REPA除了与INCI和INCⅡ重复序列外，还与REPA启动子的上游区域(可能是操纵子)以及INC II和GATC box之间的10个碱基对的部分有很强的结合。10个碱基对的区域可能是RTS1复制的起始点。ORI的分离(RTS1)。在活体实验中，当从mini-RTS1质粒反式供应REPA时，跨越1441到1194坐标的mini-RTS1亚区能够启动关联。在ORI(RTS1)中，包含了incII序列以及GATC和DNAA框。REPA的C末端缺失。构建了一系列REPA突变体，编码含有取代羧基末端6个氨基酸的寡肽的REPA衍生物。这些修饰的REPA根本不能激活ORI(RTS1)。然而，其中一个REPA衍生物对mini-RTS1保持了微弱但明显的不亲和性，这可能是由于REPA与REPA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.