| 1. The purification of SMR1The proteins SDS-PAGE analyses of the wild-type Arthrospira platensis strains Sp-S, Sp-B and Sp-T with regular helical morphology, and their corresponding linear varieties and the revertant varieties were performed. The results indicated that there were 5 significantly different bands in common between the helical and linear filaments, which were located at 53.1, 52.0, 31.8, 21.9 and 20.3 kDa, respectively. Furthermore, using the follow process and conditions, the most significant band at 21.9 kDa was purified from Sp-S. Firstly, the soluble protein extracts was extracted with Tris-HCl (pH 6.8), Secondly, in order to remove part of other proteins effectively, the crude extract was precipitated with ammonium sulfate in 40-50% saturation. Finally, the redissolved precipitate was applied to Sephacryl S-200 gel-filtration and Q-Sepharose FF anion-exchange chromatography in turn, and the electrophoresis puritied SMR1 was obtained and its yield was about 0.065%.2. The molecular characteristics of SMR1The result of 2-DE analysis showed that the pI of SMR1 was 4.98 and the purity of prepared SMR1 exceeded 90%. The secondary structure of SMR1 was investigated by Circlar Dichroism analysis, and the result revealed that the content of a-helix, β-sheet and random coil of was 31%, 12% and 57%, respectively. Furthermore, the ESI-MS analysis showed that the detected 5 polypeptides (contain 53 amino acids) belonged to CpcI, and the matched sequences were distributed in the 30th-101st amino acid. Moreover, the N-terminal sequence of SMR1 was completely matched with a section of CpcI, and the exact molecular weight of SMR1 was 16712 Da, which was determined by MALDI-TOF MS. According to the results above, it was speculated that SMR1 was a homologue of CpcI with identical amino acid sequence, and was formed by cleavage and modification of CpcI.It was reported that the nucleotide sequences of CpcI were differed withArthrospira strain. Thus the cpcHID operon in Arthrospira Sp-S was cloned and sequenced, and then the amino acid of SMRl was deduced according to the corresponding nucleiotide sequence. Than the theoretical data of molecular weight, pi and secondary structure was predicted and be compared with the experimental one. The result indicated that the two kinds of data were consistent. Moreover, it was revealed from the predicted secondary structure that SMRl domain was composed of 7 a-helix sections, whose function was involved in morphogenesis of Arthrospira, probably.3. Expression and its antibody preparation of SMRlIt was so difficult to prepare sufficient SMRl from Arthrospira for preparing antibody that we turned to express SMRl in E. coli. After studied the expression conditions, such as induced temperature and induced time, it could be concluded that the feasible temperature and time for expression of SMRl were 25-30 °C and 6-12 h respectively. Finally, the expressed product was applied to Ni-affinity chromatography for obtaining SMRl with high purity.Furthermore, the antibody against expressed SMRl was prepared by immunizing the pure-blood New Zealand rabbits for 4 times. The quantity of SMRl for every time was 1. 0.5, 0.5 and 0.5 mg, in turn. Than the antibody (antiserum) was collected in 2 weeks after the last immunization, and was determined its titer and specificity with indirect ELISA and western blot. The results indicated that the antibody against SMR1 was with a titer of 1:51200, and bound specifically to expressed SMRl and Arthrospira SMRl either. Thereby, the function of SMRl in Arthrospira morphogenesis could be revealed by some immunological methods, such as coimmunoprecipation, and protein microarry.Key words: Arthrospira platensis;morphogenesis;protein;purification;molecular characteristic;prokaryotic expression;antibody...
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