Purification And Crystallization Of Phosphorus Modified Protein DndE From Streptomyces

DNA phosphorothioation was recentlly found as the first physiological modification occurs in DNA backbone. Further research reveal that this modification can be controlled by dnd gene cluster that can encode five kind of protein that named DndA, DndB. DndC, DndD, DndE. Bioinformatics analysis suggest that DndE protein maybe a NCAIR synthesis analogue or a PAPS sulfotransferase, which make it hard to carry out research in DNA phosphorothioation. In order to clarify exact function of DndE and to study the conservative and evolutionary of DNA phosphorothioation,we chose the DndE proteins coming form Streptomyces lividans 66.Firstly we explored and optimized expression and purification of DndE protein. At frist, we try to make use of different vector and different labels, for example pET-44b、pSJ7、pET-28a. As a result, when PSMT3 vector into which dndE gene was subcloned, stable and high quality DndE protein can be obtained as DndEN-flag-livdan. After then, all DndE sample was studied on a series of functions. Research suggested that there exist many diffident conformation and existed a polymer in solution when the concentration of DndE protein increase.Because of a polymer in solution, which make it hard to solve the solution structure by NMR approach. After then we seek X-ray methods to further study the structure and function of DndE protein. Firstly we carried out crystal screen, and then develop a series of crystallization screening and optimization of crystallization. When we carry on crystal screening, we get a lot of crystal of the DndE. Then, we optimize the condition of a series of good crystals. Unfortunately, we have not get the crystal structure of DndE because short of the crystal analysis resolution.According to recent reports, it finds that DndE protein form Escherichia coli B7A may be a novel DNA binding protein. So, we also feel that DndEN-flag-livdan may be the same. In order to prove the viewpoint. We developed random DNA stranded molecule which contains label of fluorescent to carry out research. It was found that random DNA stranded have has stronger binding with the protein of DndE. So we speculated that DndE protein has non-specific binding affinity with DNA, is the novel DNA binding protein. In the whole mechanisms of DNA sulfur modification, maybe play the role of a transference.