Purification of Rts1 RepA protein and its characterization

Rts1 RepA 蛋白的纯化及其表征

• 批准号: 61480147
• 负责人: TERAWAKI Yoshiro
• 金额: $ 4.29万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61480147/
• 关键词: plasmid Rts1; DNA replication; replication protein; purified RepA protein; DNA binding protein; DNase I foot print; 変異RepA; 複製調節; DNaseIフットプリント法

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.

1.RepA蛋白的纯化。将编码Rts1复制所必需的RepA蛋白的repA基因插入载体质粒pKK223-3中，从而在大肠杆菌JM103中在tac启动子的控制下表达repA。使用 CM-sephadex、磷酸纤维素和 Affigel blue 柱通过色谱法纯化从 IPTC 诱导的 10 升细胞培养物中获得的粗裂解液中的 RepA。纯化后的RepA的氨基酸组成与repA序列推导的氨基酸组成一致。 2． RepA 与 DNA 的结合（通过 DNase I 保护测定进行检查）。纯化的 RepA 被证实与 repA 启动子的紧邻上游区域（这将是一个操纵子）以及 incII 和 GATC 框之间的 10 bp 部分强烈结合incI 和 incII 重复序列。 10 bp 区域可能是 Rts1 复制的起始位点。3．分离ori(Rts1)。当体内研究中从 mini-Rts1 质粒反式提供 RepA 时，跨越坐标 1441 至 1194 的 mini-Rts1 亚区能够启动复制。在ori(Rts1)中，包含incII序列以及GATC和DnaA框。4． RepA 的 C 末端删除。构建了一系列repA突变体，其编码含有寡肽取代取代羧基末端六个氨基酸的RepA衍生物。这些修饰的 RepA 根本无法激活 ori(Rts1)。然而，其中一种 RepA 衍生物与 mini-Rts1 质粒保持微弱但明显的不相容性，这可能是由于 RepA 与 repA 操纵子的结合引起的。

项目成果

伊藤義文: 日本細菌学雑誌. 42. 250 (1987)

伊藤义文：日本细菌学杂志 42. 250 (1987)。

Yoshiro,Terawaki: "Importance of the C terminus of plasmid Rts1 RepA protein for replication and incompatibility of the plasmid" J. Bacteriology. 170. (1988)

Yoshiro,Terawaki：“质粒 Rts1 RepA 蛋白的 C 末端对于质粒复制和不相容性的重要性”J. Bacteriology。

伊藤義文: Journal of Bacteriology. 169. (1987)

伊藤义文：细菌学杂志 169。（1987）

神尾好是: J. Bacteriology.

Yoshize Kamio：J.细菌学。

Yoshifumi,Itoh: "Essential DNA sequence for the replication of Rts1" J. Bacteriology. 169. 1153-1160 (1987)

Yoshifumi,Itoh：“Rts1 复制的基本 DNA 序列”J.细菌学。