Signal transaction between the functional domains of type II restriction endonuclease

StsI是一类不寻常的限制性内切酶的成员，它识别特定的DNA序列并在距离该序列较短的距离处非特异性地切割。本研究描述了限制性内切酶StsI失去DNA切割活性的突变体。我们构建了不能特异性切割DNA的StsI变体S233I、W241R、K300E、L365R、E442K、L453S和I478T。突变体S233I和E442K从重组大肠杆菌细胞的可溶性部分均质纯化，但其他突变体酶产生包涵体。为了从包涵体中纯化这些蛋白质，将聚集体悬浮在6 M胍- hcl中，并用适当的缓冲液稀释后重新折叠。突变蛋白随后用肝素- sepharose层析纯化。StsI的变体L365R在DNA结合和DNA切割活性上都存在缺陷，它可以刺激野生型StsI催化的DNA切割速率。这些结果表明，StsI催化结构域是二聚的，正如FokI报道的那样，但第二个StsI分子不一定与识别位点结合。为了解决StsI的晶体结构，构建了过表达系统，并对酶进行了均质纯化。然而，我们还没有成功地获得StsI晶体。StsI的净化工艺有待改进。

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.