Study On The Cloning, Expression And Purification Of Ciona Savignyi Polypeptide CS5931 And Its Mode Of Action Of Antitumor Activity

Previous study in our laboratory has shown that a novel polypeptide, CS5931 derived from C iona savignyi possesses potent antitumor activity and its molecular weight is 5931 Da. CS5931 inhibited the growth of many kinds of tumor cells and induced apoptosis in human hepatoma cell line BEL-7402 in vitro. N-terminal amino acid sequence determination of CS5931 indicated that the polypeptide displays high homology with the sequence of human granulin(GRN). In the present study, the full length cDN A of CS5931 precursor was cloned by RACE(rapid amplification of cDNA ends) approach and the amino acid sequence of CS5931 was obtained, and the polypeptide was expressed in E. coli with high efficiency. The expressed protein was purified through affinity chromatography. The purified polypeptide displays antitumor activity identical with that of native CS5931. The antitumor activity and molecular mechanism of recombinant CS5931 were also presented.The full length c DNA of CS5931 precursor, termed Cs-pgrn-1 was cloned by through RACE. The complete cDN A sequence of this gene consists of 685 bp containing an open reading frame(ORF) of 522 bp(173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin(GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares the highest homology with Ciona intestinalis GRN and shares high similarity with human GRN A, B and C. This result suggests that CS5931 is conserved during evolution. In addition, prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A(RSM=1.54?).The gene fragment coding CS5931 was successfully cloned and then linked into prokaryotic expression carrier p ET32a(+) and p ET21a(+), respectively. Two expression vectors p ET32a(+)-CS5931 and p ET21a(+)-CS5931 were transformed into E. coli Origami(DE3)without or with chaperone plasmid p G-KJE8 to construct protein expression system Origami/p ET32a-CS5931 and Origami/p ET21aCS5931+pG-KJE8, respectively. Then fusion protein Trx-CS5931 with thioredoxin tag and recombinant polypeptide CS5931-His were induced and expressed successfully in soluble form using Origami/pET32a-CS5931 and Origami/p ET21aCS5931+pG-KJE8, respectively. This illustrated that both thioredoxin and molecular chaperones can promote formation of intramolecular disulfide bonds in CS5931. After denaturation, purification, renaturation and digestion with protease, the recombinant polypeptide rCS5931 was obtained with purity over 95%. And at the same time, we also obtained the recombinant polypeptide CS5931-His with His-tag and its purity was above 95%. Recombinant polypeptide rCS5931 and CS5931-His both exhibited inhibitory activities to human cervical carcinoma Hela cells with IC50 values of 2.74μM and 3.17μM, respectively. The recovery rates of rCS5931 and CS5931-His were 1.8% and 28.2%, respectively. Thus expression system Origami/p ET21a-CS5931+pG-KJE8 is especially adapted for the large-scale the recombinant polypeptide purification.Antitumor effect of CS5931-His was studied by analysis of cytotoxicity activity of tumor cells in vitro by MTT assay. The results showed that CS5931-His significantly inhibited the growth of the selected five cells, including human cervical carcinoma Hela cells, human colon carcinoma HCT116, RKO cells, human A549 lung carcinoma cells, human leukemia HL60 cells, with IC50 values of 3.17, 2.00, 5.14, 4.29 and 4.78μM, respectively.. This result suggested that human colon carcinoma HCT116 cells are the most sensitive to the treatment of CS5931-His. After treatment with CS5931-His at the concentrations of 4μM and 8μM, the typical apoptosis was observed in HC T116 cells by the result of Annexin V-FITC/PI double staining confirmed that CS5931-His induced apoptosis in HC T116 cells in a dose-dependent manner.Immunocytofluorescen analysis showed that CS5931-His was mainly located in cell membrane and interacted with membrane-associated protein. Further study showed that CS5931-His interacted with human Enolase 1 as determined by His-pull down and immunoprecipitation assay. To further confirm the interaction between CS5931-His and Enolase 1, co-immunoprecipitation and double-labelling immunofluorescence were performed and the interaction was certified. However, it is not sure that the inhibition of Enolase 1 activity is the key mechanism for the anti-tumor effect of CS5931 and more experiments will be performed to confirm it. The study is important for the development of the polypeptide as novel anticancer agent. The study also provides insight into the understanding of the function of Enolase 1 as a novel target in cancer treatment.