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

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。

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

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.