Signal transaction between the functional domains of type II restriction endonuclease

II型限制性内切酶功能域之间的信号交换

• 批准号: 11660092
• 负责人: KITA Keiko
• 金额: $ 2.43万
• 依托单位: TOTTORI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660092/
• 关键词: Restriction endonuclease; Functional domain; Overexpression; Fusion protein; DNA-binding protein; Chimeric enzyme; Crystallization; Folding; DNA結合活性; 人工酵素; 高発現ベクター; アフィニティーカラム; インクルージョンボディー; リフォールディング; ランダム変異; エラー・プローン PCR

StsI is a member of unusual class of restriction enzymes that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence. The present study describes mutants of the restriction enzyme StsI that lose DNA cleavage activity. We constructed StsI variants, S233I, W241R, K300E, L365R, E442K, L453S, and I478T, which are unable to cleave DNA specifically. The variants, S233I and E442K, were purified homogeneously from the soluble fraction of recombinant E. coli cell, but the other mutant enzymes produced inclusion bodies. In order to purify these proteins from inclusion bodies, the aggregates were suspended in 6 M guanidine-HCl, and refolded by dilution with the appropriate buffer. The mutant proteins were purified homogeneously with subsequent chromatography using Heparin-Sepharose. The StsI variant, L365R, which defective for both DNA-binding and DNA-cleavage activities, stimulated the rate of DNA cleavage catalyzed by wild-type StsI. These results suggested that the StsI catalytic domains dimerize, as reported by FokI, but a second StsI molecule does not necessarily bind to the recognition site.In order to solve the crystal structure of StsI, an overexpression system was constructed and the enzyme was purified homogeneously. However, we have not been succeeded in obtaining the StsI crystal yet. Improvements have to be made in purification process of StsI.

StsI是一种不寻常的限制性内切酶，它能识别特定的DNA序列，并在离该序列很短的距离内进行非特异性切割。本研究描述了突变体的限制性内切酶StsI，失去DNA切割活性。我们构建了StsI变体S233 I、W241 R、K300 E、L365 R、E442 K、L453 S和I478 T，它们不能特异性切割DNA。从重组E.大肠杆菌中表达，而其他突变体产生包涵体。为了从包涵体中纯化这些蛋白质，将聚集体悬浮在6 M盐酸胍中，并通过用适当的缓冲液稀释来重折叠。突变体蛋白用随后的层析使用肝素-琼脂糖凝胶进行均匀纯化。StsI变体L365 R，其DNA结合和DNA切割活性都有缺陷，刺激了野生型StsI催化的DNA切割速率。这些结果表明，StsI催化结构域的二聚体，如报告的FokI，但第二个StsI分子不一定结合到识别位点。然而，我们还没有成功地获得StsI晶体。StsI的纯化工艺有待改进。