Huang B, Schaeffer C J, Li Q, Tsai M D
Ohio State Biochemistry Program, Ohio State University, Columbus 43210, USA.
J Protein Chem. 1996 Jul;15(5):481-9. doi: 10.1007/BF01886856.
A new restriction endonuclease, named Splase, was constructed by genetically fusing the DNA-cleavage domain of the restriction endonuclease Fok1 with the zinc-finger DNA-binding domain of the transcription factor Sp1. The resulting protein was expressed in Escherichia coli., partially purified, and shown to selectively digest plasmid DNA harboring consensus Sp1 sites. Splase was also shown to selectively digest the long terminal repeat of the HIV-1 DNA at Sp1 sites. Splase recognizes a 10-bp DNA sequence and hydrolyzes phosphodiester bonds upstream of the binding sequence. The binding specificity of Splase makes this a "rare cutter" restriction enzyme which could be valuable in creating large DNA fragments for genome sequencing projects. The result also presents the opportunity to create other restriction enzymes by altering the binding specificity of the zinc-finger recognition helix.
一种名为Splase的新型限制性内切酶是通过将限制性内切酶Fok1的DNA切割结构域与转录因子Sp1的锌指DNA结合结构域进行基因融合构建而成的。所得蛋白质在大肠杆菌中表达、部分纯化，并显示出能选择性地消化含有共有Sp1位点的质粒DNA。Splase还显示出能在Sp1位点选择性地消化HIV-1 DNA的长末端重复序列。Splase识别一个10个碱基对的DNA序列，并在结合序列上游水解磷酸二酯键。Splase的结合特异性使其成为一种“稀有切割”限制性酶，这对于为基因组测序项目创建大的DNA片段可能具有重要价值。该结果还提供了通过改变锌指识别螺旋的结合特异性来创建其他限制性酶的机会。
