Expression, Purification And Spectroscopic Analysis Of Euplotes Octocariratus Centrin

Centrins play a fundamental role in centrosome duplication and contraction of centrin-based fiber systems in eukaryotic cells. Up to now, the studies on the structure and function of Centrins have still been one of the popular subjects in Cell Biology. However, the studies of this sort, at home and aboard, are mainly focused on biological function, and its X-ray crystal structure has not been reported yet. Besides, researches on the relation between structure and function from the perspective of solution structure are rare as well.In recent years, with the exploitation of rare earth resources, this group of biologically unessential elements is becoming closely related with people's daily life. Therefore, it is imperative for us to understand the mechanism of rare earth in living systems and its long-term effects on organisms. Centrins are a subfamily within the superfamily of Ca2+-modulated proteins. It contains four potential Ca2+ binding sites, known as EF-hand motif. So we infer that Centrins will be the carrier by which rare earth ions achieve their biological effect in life-body.Till now, we have not found any report on the expression of unicellular lower eukarya Euplotes Centrin, solution conformation and biological function. In this work, expression plasmids pGEX-6p-l-centrin were constructed by means of PCR and recombinant DNA techniques. Fusion expression of Centrin in E, coil BL21was performed by induction of IPTG Fusion protein was cut by PPase and was purified by GST chelating affinity chromatography and ion exchange chromatography. The final products were checked by SDS-PAGE.Some parameters and properties of Centrin, after being purified, were predicted with GenePro, such as isoelectric point, net charge, amphipathy, hydropathy, a-helix potential, Calcium-binding site. All these attempts aim to provide some theoretical guidance for the succeeding experiential researches. The analysis shows that Centrin is an acidic, low molecular calcium-binding protein and contains four EF-hand helix-loop-helix motifs, one Ca2+ binded in each of motifs. However, the binding capacities to calcium at N-termial and C-terminal lobes are likely to be different.Then its interaction with metal ions and a model peptide is investigated by UVand fluorescence spectra technique. The results demonstrate that upon the combining of Ca2+ or Tb3+ with apoCentrin, the binding sites are same and in a same sequence, i.e. the III and the IV sites at C-terminal lobe are preferably occupied. When protein is combined with metal ions, the sidechains of aspartic acid and glutamic acid residue at the four Ca2+ binding sites of protein participate in the coordination of metal ions. Tb3+ is able to contend for the Ca2+-binding sites and occupy them completely. The existence of Ca2+ does not influence the binding sequence of Tb3+ to Centrin at all.The interaction between Melittin and Centrin, not depending on Ca2+, forms 1:1 complexes and the solution structure of Centrin after this interaction may change from dumpbell shape to ball shape. The binding of Tb3+ with apoCentrin takes no effect on the interaction between protein and Melittin, while the interaction between Melittin and apoCentrin causes that Tb3+ is unable to be binded to protein. Upon the interaction of Melittin with Centrin in the presence of Ca2+, Tb3+ can be binded to Centrin but cannot occupy its four binding sites completely.