Cloning, Expression And Purification Of Escherichi Coli DNA-binding Protein And Study On Its Self-assembly In Vitro

Dps（DNA-binding protein from starved cells）proteins,which exist widely in archaeaand bacteria and belong to Ferritin superfamily, play an important role in stress protection.They can make response to oxidative stress by means of the combination with iron and itsferrous iron oxidase activity and then result in the oxidation and storage of iron so as toeliminate the toxicity of H2O2and Fe(Ⅱ) and effectively protect DNA, protein andmembrane lipid from oxidative damage. In addition, under some stress conditions such asiron, copper, radiation, high concentrations of salts, acid and base shock, starvation,nutrient deprivation and so on, a variety of bacterial Dps proteins are capable of bindingDNA in a sequence-independent manner and forming the highly ordered and stableDps-DNA complexes for the transformation of chromosome structure from the easilydamaged loose state to the agglutinative and compact state to protect DNA from thedamage by various stress factors. Dps proteins can self-assemble into dodecamers,however, the machanism of the process has been having a smattering of knowledge. Hence,the deep focusing on the self-assembly mechanism of Dps proteins and the effectiveconditions on polymerization and depolymerization will contribute to the explanation ofDps functions and its application in biological nanotechnology process and immunetherapy.In this work, the Dps gene from Eschericha coli was cloned by PCR technique usingE coli genome as a template and was added with a His tag on its downstream, and then wasrecombinated into pBV220vector and pBV-Dps-6His prokaryotic expression vector wassuccessfully constructed. Further, two mutant expression vectors of pBV-Dps-2X-6His andpBV-Dps-Ua-Ub-6His encoding mutant Dps proteins were constructed with the similarprocess. The Dps-2X-6His protein was a mutant which five amino acids were inserted intoDps-6His between Y107and P108. The Dps-Ua-Ub-6His was another mutant which wasinserted into Dps-6His between Y107and P108by the47amino acid sequence that coulddisplay multi-epitope antigen activity of Helicobacter pylori urease A and B subunits. By42℃temperature induction, Dps-6His, Dps-2X-6His and Dps-Ua-Ub-6His proteins werehighly expressed in prokaryotic expression cells(strain DH5α) with the forms of inclusionbody. The three proteins were purified by affinity chromatography and iedntified byWestern Blotting respectively. The data from in vitro experiments showed that Dps-6His proteins could self-assemble into dodecamer and the process was affected by Na+, Mg2+and SDS. However, Dps-2X-6His and Dps-Ua-Ub-6His proteins were easy todepolymerize instead of polymerize. It was suggested that the mutant Dps with insertion ofexogenous sequence had a huge influence on both the structure and the self-assemblefunction of Dps protein. Dps-6His protein was able to bind DNA in asequence-independent way and it was preliminary speculated that this combination relieson the concentration of Dps-6His dodecamer proteins. Dps-6His proteins could also protectplasmid DNA from reactive oxygen species oxidative damage and the very strongprotection was aquired with only trace proteins. The interaction in some way betweennanosilver particle (Np-Ag) and Dps-6His protein could take place and might be related tothe sructure of the protein as the stronger interaction was shown in natural Dps protein andthan denatural one. The results of SDS-PAGE indicated that Dps proteins and iron couldcombine in a certain manner and form nanoscale iron particles in vitro.