Expression, Purification And Crystallization Of DndB Protein Involved In DNA Phosphorothioation

DNA sulfur modification is a new DNA modification phenomenon found in recent years, which is widespread among prokaryotes. It is determined by five genes-dndA, dndB, dndC, dndD and dndE, which locate in one gene cluster and code five corresponding proteins(DndA, DndB, DndC, DndD, DndE) respectively. This study discussed the expression, purification and crystallization of DndB, the expression vectors construction of different DndB fragments and the purification of DndA and DndE.DndB protein in Salmonella enterica serovar Cerro 87 consists of 361 amino acid residues with a relative molecular weight of 41 kDa. DndB showed significant amino acid sequence homology to a group of transcriptional regulators, it seemed to bind with DNA to determine the sulfur modification specificity. In this study, we purified enough DndB protein and got its crystal. But the crystal was not good enough to conduct X-Ray diffraction. After optimization, its shape and size were improved, while still not qualified enough to X-Ray diffraction.During research, we found that Salm-DndB's crystal degraded into two different bands, and the big band's sequence proved to be 172-361 fragment whereas the small band's sequence was not confirmed, and we used 1-171 to replace it, so we constructed the expression vectors of Salm-DndB 1-171 and Salm-Dnd B 172-361. Unluckily, we did not get their soluble proteins. Meanwhile, according to Salm-DndB FoldIndex folding forecast, we constructed the expression vectors of Salm-DndB 1-312 and Salm-DndB 108-361 fragments and according to Salm-DndB sequence conservation, we constructed the expression vector of Salm-DndB 54-361 respectively, But Salm-Dnd B 1-312 didn't get expressed, Salm-DndB 54-361 appeared to be precipitated and only Salm-DndB 108-361 got expressed with a small amount.In this study, we also purified DndA protein in Streptomyces lividans 66, namely, Strep-DndA for further use. As to DndE protein, we purified B7A-DndE protein of E.coli B7 A and got B7A-DndE with high purity after molecular sieve chromatography, but the protein uniformity is not good enough.