Optimization Of Fermentation And Purification And The Antitumor Activity Of Polypeptide From Ciona Savignyi

Ciona, limited the development of aquaculture, were widely distributed in the coastal area of Shandong province. Recently, some studies found that Ciona have some biological actions such as antitumor, antivirus, antimicrobial, immunity adjustment and biocatalysis. Especially notable is the antitumor, antioxidant effects and the function for the growth of brain. In a word, Ciona have good application prospects.A novel polypeptide CS5931, derived from Ciona savignyi possesses potent antitumor activity. N-terminal amino acid sequence determination of CS5931 indicated that the polypeptide displays high homology with the sequence of human granulin (GRN). Previous study in our laboratory has constructed the recombinant strains of CS5931. Expressed CS5931 inhibited the growth of many kinds of tumor cells.The optimal of fermentation conditions is an important process to improve the volume of polypeptide for recombinant Escherichia coli expression system. The fermentation process of recombinant CS5931 expression in Escherichia coli BL21(DE3)/pET21a-CS5931 was studied. The effects of factors such as concentration of IPTG, IPTG add time, induction time, inoculum quantity, loading volume, induction temperature were investigated. Plackett-Burman and Central-Composite design were developed based on one factor tests. By Optimizing the fermentation of expressed CS5931 in Escherichia coli, the yield of CS5931changed to 0.703 g/L. It was 2.01 times better than before.Prepare the bacterial sample by super centrifugation and sonication. Purify the CS5931 by Ni-NTA with AKTA-Purifier. By changing the kinds of buffer, the concentration of hydrosaline solution, the way of renaturation and the freeze-drying technology, the yield of refolded CS5931 was above 30% and achieved the set.Antitumor effect of expressed CS5931 was studied by analysis of cytotoxicity activity of tumor cells in vitro by MTT assay. The results showed that the expressed CS5931 significantly inhibited the growth of the selected four cells, including HCT116 human colon carcinoma cells, Panc28 human pancreatic carcinoma cells, NCI-H460 human large cell lung carcinoma cells, BEL-7402 human hepatoma cells, with IC50 values of 1.41,2.04,4.28,11.71 μM. However, CS5931 exhibited low cytotoxicity against HFL1 human normal fibroblast lung cells.By optimizing the fermentation process and the technology, the yield of the recombinant polypeptide CS5931 is improved. CS5931 has good inhibitory activity on the tumor cells. This study is important for further pilot amplification and pharmacology, pharmacodynamics and toxicology research.