Expression, Purification And Characterization Of DndA In Streptomyces Lividans

DNA is an important carrier of genetic material, DNA modification generally occurs in the composition of DNA guanine, adenine, cytosine, thymine. The phosphorothioation DNA modification occurs between the phosphorus atoms and oxygen atom of DNA backbone. Thus the research of DNA phosphorothioation modification is of great significance. The early studies showed that the DNA phosphorothioation modification is related with five proteins which encoded by dnd gene cluster(dndA-E). In which, dndA gene encoding the protein DndA. In 2012, the team of Deng Zixin resolved the the crystal structure of DndA(C/S) protein. Compared with other reported cysteine desulfurases, the crystal structure of DndA(C/S) is different. The active site Cys327 of DndA(C/S) is located on a ? fold,but traditional active site cysteine located on a flexible loop.Is this structure diffenence caused by the active site mutation or DndA has a new desulfurization mechanism?In order to study the structure of DndA(WT), we successfully constructed dndA(WT)and mutant dndA(C/S) plasmids and made a plasmid expressing solubility of the supernatant test; We purified the protein of DndA(WT) and DndA(C/S) by nickel column chromatography, anion exchange chromatography and gel filtration chromatography. The purified protein reached 95% both DndA(WT) and DndA(C/S). CD spectra and analytical surperdex 75 showed there is no substantial structure difference between DndA(WT) and DndA(C/S).In order to investigate the structure difference and the detailed mechanism of desulfurization. We constructed a series of plasmids needed in segmental isotopic labeling for DndA and purified the proteins. In the case of DndA PTS method not achieving the desired effect, we reinvented the plasmid in PTS method for the later protein detection. At the same time, we tried the protein ligation of N-terminal and C-terminal without the aid of a small peptide.