Clam Polypeptide Cloning, Expression, Purification And Anticancer Activity

Past study in our laboratory has shown that Mere15 were extracted from Meretrix Meretrix possesses anticancer actvities and its molecular weight is about 15 kDa. Mere15 inhibited the cancer proliferation and boost cancer cell programmed death. N-terminal amino acid sequence determination of Mere15 indicted that the protein displays high homology with the sequence of Meretrix Meretrix tropomyosin. In the recent study, the tropomyosin have been investigated for many years, but the anticancer activities have not mention. We found the peptide own effective anticancer activities. The full length cDNA of was clone by RACE(rapid amplification of cDNA ends) approach and the amino acid of tropomysoin was achieve, and the protein was expressed by E. coli with His-Tag. The peptide was purified by affinity chromatography. The recombination peptide could evidently inhibited cancer cell proliferation.We concreted clams tropomyosin expression system. According to tropomyosin gene, we found a dozen different bases in the sequence which amplified by gene cloneing. But protein is as same as clams polypeptide. The reason is not very clearor may be E. coli codon system is different with eukaryotes creatures. The engineering bacteria were induced by IPTG to screcte the recombinated protein. But the production was very insufficient, so we design an other experiment to improve the prodution.Tropomyosin Prokaryote expressionsystem was optimized by response surface method. Firstly, Plackett-Burman design was applied to evaluate ten fermentation factors. With statistic analyses, the signification factors effect was determined as following: glucose, yeast extract a nd magnesium sulfate. And then, CCD(central composition design) was used to gauge the three factors optimization proportion. By the regression quadratic model equation analysis, the conclusion is that optimal concertation of the three factors were determined as glucose 10.41 g/L, extract yeast 11.37g/L and magnesium sulfate 0.44 g/L, and the protein prodution was promoted to 156.3%. The confirmatory test condition was as same as the predicted valus, and the number indicated that the model was suit and effective for experiment.Mere15 anticancerv activities have been studied by analysis of cytotoxicity activity of tumor cells in vitro by MTT assay. The results showed that tropomyosin-His significantly inhibited the growth of the selected two cells, including human A549 lung carcinoma cells and human Pc28 cells, with inhibition rate 46.17 and 47.78 %.. By DAPI staining, we compared different concentration of protein results in the cells research. The results show that drugs on cells have a good inhibition at high concentrations which may well induce cancer cell apoptosis. The results showed that the simple drug made cells dyeing by DAPI is brighter than control group. From low to high drug concentration on cells, the drug activity exhibited a certain concentration gradient in lung cancer cells and ovarian cancer cells, indicated that the effect of the drug is relatively good.