Effects Of Site Direct Mutation On The Characterization Of Euplotes Octocarinatus Centrin

Centrin is a member of the calcium-binding EF-hand protein superfamily present in centrosomes of widely divergent species. It was originally cloned at the DNA level from the unicellular green alga Chlamydomonas reinhardtii. It has been found to be localized to the major microtubule organizing center in its respective cells. There is also evidence to suggest that it is required for the normal duplication and segregation of the microtubule organizing center. Like calmodulin and troponin C, caltractin contains four potential calcium-binding sites of predicted helix-loop-helix or EF-hand structure.With the development of technology in biology, more and more academicians study questions for discussion by mutating genes in organisms. In the work, we got mutated plasmids pGEX-6P-M-Centrein by means of PCR and recombinant DNA techniques. Fusion expression of M-Centrin in E.Coil BL21 was performed by induction of fPTG. Fusion protein was digested by PPase and was purified by GST chelating affinity chromatography and ion exchange chromatography. The final products were checked by SDS-PAGE gel.We got some significant results regarding M-Centrin by chemical methods. The titration of M-Centrin by terbium ions was monitored by UV different spectrum and fluorescence spectrum. Mutant gene affected M-Centrin propertities. From UV different spectrum, we know that there are fine absorption of Tyr and Trp apart from those of Phe. The wavelength of protein at which the fluorescence intensity of protein was maximal is 360nm but not 310nm. There is evidence that suggest M-Centrin contains four EF-hand helix-loop-helix motifs. Moreover the binding capacities to metal ions at N-terminai and C-terminal lobes are different. In the context, the forth and third EF-hands are superior to the rest EF-hands in the capabilities of combining terbium ions with protein. Moreover we have got the macroscopic binding constants of KTb-Pro by monitoring fluorescence. Independent of calcium ions, protein can also combine with Melittin. We can get thesame result when calcium ions is replaced by terbium ions.