Signal transaction between the functional domains of type II restriction endonuclease

STSI是一种特殊的限制性内切酶，可以识别特定的DNA序列，并在距离该序列很短的距离内进行非特异性切割。本研究描述了失去DNA切割活性的限制性内切酶STSI的突变体。我们构建了不能特异性切割DNA的STSI突变体S233I、W241R、K300E、L365R、E442K、L453S和I478T。突变体S233I和E442K从重组大肠杆菌的可溶性部分中得到了均一的纯化，而其他突变酶产生了包涵体。为了从包涵体中纯化这些蛋白，将聚集体悬浮在6M盐酸胍中，并用适当的缓冲液稀释复性。用肝素-琼脂糖柱层析对突变蛋白进行均一纯化。同时缺乏DNA结合和DNA切割活性的STSI变异体L365R刺激了野生型STSI催化的DNA切割速度。这些结果表明，正如FokI所报道的那样，STSI催化结构域是二聚的，但第二个STSI分子并不一定与识别位点结合。为了解决STSI的晶体结构问题，构建了一个高效表达系统，并对该酶进行了纯化。然而，我们还没有成功地获得STSI晶体。STSI的提纯工艺有待改进。

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.