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

摘要:

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。

Abstract:

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.