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

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.