Preparation of restriction endonuclease Mlu I and its seleno-derivative

Abstract:

[Background] Restriction endonuclease Mlu I is a commonly used tool in molecular biology. However, its three-dimensional structure has not been determined. [Objective] Mlu I gene was cloned and expressed in Escherichia coli. Mlu I and its seleno-derivative proteins were purified, and the crystallization conditions were studied. [Methods] A recombinant expression vector pET28b-Mlu I was constructed and expressed inductively in E. coli BL21(DE3)pLysS. Affinity and gel filtration chromatography were used to purify Mlu I and Se-Mlu I proteins. Mass spectrometry, circular dichroism and enzyme activity analysis were carried out to characterize these proteins. Sitting drop method was used to screen the crystallization conditions. [Results] The recombinant expression vector pET28b-Mlu I was successfully constructed and the purified Mlu I and Se-Mlu I proteins with purity suitable for crystallization were obtained. All 8 methionines were successfully replaced with se-methionines in the Se-Mlu I protein determined by mass spectrometry. The circular dichroism and enzyme activity analysis confirmed that se-methionines replacement had no significant effect on the activity and structure of the Mlu I protein. Preliminary crystallization study and subsequent X-ray diffraction showed that the needle-like crystal of Mlu I formed under one condition diffracted to a resolution of 0.32 nm. [Conclusion] The construction of Mlu I and Se-Mlu I protein purification system and study of crystallization conditions layed the foundation for further analysis of the three-dimensional structure of Mlu I, revealing the molecular mechanism of Mlu I, and directed evolutionary modification of Mlu I.