限制性内切酶NcoⅠ的高效重组表达、硒代与结晶条件初步筛选

High Efficient Recombinant Expression of Endonuclease NcoⅠ Selenoprotein and Preliminary Screening of Protein Crystallization Conditions

来源于珊瑚诺卡氏菌Nocardia corallina的限制性内切酶NcoⅠ是基因工程中常用的工具酶之一。然而目前NcoⅠ蛋白质三维结构尚未被解析,对其基因改造缺乏理论指导。为了解析NcoⅠ的三维结构,采用大肠杆菌表达系统,高效重组表达和纯化,获得了纯度>95%的NcoⅠ野生型和Se-NcoⅠ硒代蛋白。质谱分析表明,重组Se-NcoⅠ蛋白中所有硫原子成功被硒原子取代。酶切实验表明,硒代蛋白与野生型具有相似的限制酶活性。采用坐滴法筛选重组NcoⅠ蛋白结晶条件,已在3种条件下获得针状晶体,1种条件下获得颗粒状晶体。X射线衍射初步分析晶体分辨率在0.8 nm左右,为下一步解析NcoⅠ三维结构提供了基础。

Type Ⅱ restriction endonuclease NcoⅠ derived from Nocardia corallina is one of the enzymes commonly used in genetic engineering. However, the three-dimensional structure of NcoⅠ has not been determined, and there is no theoretical guidance for the genetic modification of NcoⅠ. To analyze the crystal structure of NcoⅠ, wild-type NNcoⅠ and its selenoprotein Se-NcoⅠ with purity >95% were obtained by high efficient recombinant expression and purification using E. coli system.Mass spectrometric analysis showed that all sulfur atoms in Se-NcoⅠ were successfully substituted by selenium atoms. Enzyme digestion experiments showed that selenoproteins had similar restriction enzyme activity with the wild-type. The crystallization conditions of wild type NcoⅠ were screened by sitting-drop method. Needle-like crystals were grown in three conditions and granular crystals were grown in another condition. A set of data with a resolution of 0.8 nm was collected by preliminary X-ray diffraction analysis, which provided a basis for further analysis of the three-dimensional structure of NcoⅠ.