Study On The Centrin Of Euplotes Octocarinatus

The number, direction and polarity of microtubules in eukaryotic cells are usually organized by microtubule-organizing centre (MTOC). Alteration of the MTOC duplication could severely perturb the bipolar spindle formation, and this blocks the cell division in the G2/M phase. In contrast, hyper amplification of the centrosome is associated with cellular transformation and cancer. About 150-200 proteins are permanently or temporarily part of this matrix and among them are several regulatory proteins such as centrin. Centrin is an acidic protein of 19.5 kDa, which belongs to the highly conserved EF-hand CaM super family of Ca²⁺-binding proteins. It is a ubiquitous highly conserved protein in diverse evolutionary lineages, including algal, higher plants, invertebrate and mammalian cells. The high degree of conservation and the ubiquity of the protein in all species investigated so far suggest that centrin is essential for proper cell function. It has been shown to be required for the normal duplication and separation of the microtubule-organizing center. Mutations and complete deletions of the centrin gene have shown that the protein is required for proper cell division. It also plays fundamental roles in contraction of centrin-based fiber systems in eukaryotic cells.Euplotes octocarinatus, a unicellular protozoa, locate in a special phylogenic degree showing a number of exceptional features. MTOC of the unicellular protozoa ciliate is not structurally defined. In Euplotes, the microtubules were organized by the microtubule-organizing center of karyolymph. In this study, the centrin gene was cloned from Euplotes octocarinatus using degenerated primers and telomeric primers. The results of sequence analysis indicated that the full-length gene contains 690 bp including one open reading frame (507 bp) without intron and two telomeric sequences C4A4C4A4C4A4C4 on the termini of the gene. A full length and a N-terminal truncated fragment of the centrin gene were ligated into the expression plasmid pGEX-6P. The fusion proteins GST-EoCen andGST-EoCenN were over-expressed in an E.coli strain B121(DE3). The proteins were purified to above 95% of purity by GST affinity chromatography and ion exchange chromatography Mono Q and pure protein about 10 mg per liter LB medium were obtained.The aggregation characterization of EoCen was examined by size exclusion in vitro. We found that ion strength had an important impact on the protein's aggregation and most EoCen showed elongated monomer when salt was presence. The protein without NaCl had the tendency to produce more aggregation with increased basic concentration. The aggregation of the protein in NaCl solution did not change greatly when Ca2+ was added. If put the Ca2+ into the protein solution without NaCl, the protein would precipitate until the NaCl was added and this process was reversible. A truncated form of EoCen, lacking the first 23 residues, shows no turbidity.The results from UV and fluorescence spectra showed that all four EF-hand motives could bind Ca2+ and the III and IV sites at C-terminal lobe were preferably occupied. The CD spectrum did not change much when the temperature and pH changed and this indicated that EoCen was a stable protein.Good quality, well diffracting crystals could be obtained from a condition containing 0.2M NaCl, 20%PEG 3350 and the protein was dissolved in 10 mM CaCl2- EoCen crystals belong to space group $2\2{l\ and diffract to 2.01 A. The unit-cell parameters are a=34.4 A, b=48.9A, c=72.6 A (Fig.2). Assuming the presence of one molecule in the asymmetric unit, the solvent content is estimated to be about 19.4% and the Matthews coefficient (VM) is about 1.5 A3 Da"1 o All the results indicated that the NaCl and Ca2+ were important for the stability of the EoCen.Wistar rats were immunolized by the purified EoCen with the injection dosage of 0.4ug antigen/g body weight. The polyclonal antibody was prepared. The antibody titer of 1:2560 was detected by indirect ELISA. The antiserum was purified using DEAE-dextran and PVDF membrane. The total proteins from Euplotes was detected by Western blotting with anti-EoCen...