Abstract 1508: Cloning, expression and purification of Cionasavignyi polypeptide CS5931 and its antitumor activity

Abstract

Abstract Previous study in our laboratory has shown that a novel polypeptide CS5931 (molecular weight 5931 Da) derived from Ciona savignyi possesses potent cytotoxicity against a variety of human cancer cells including cervical carcinoma Hela cells, colorectal carcinoma HCT-116 and RKO cells, lung carcinoma A549 cells, and leukemia HL60 cells ,with IC50 values of 4.17, 2.50, 5.54, 4.69 and 4.98uM,respectively. This result suggested that HCT116 cells are the most sensitive to the treatment of CS5931. We also found that CS5931 markedly induced apoptosis in a dose-dependent manner in HCT116 cells by Annexin V-FITC/PI double staining analysis. However, its content in Ciona savignyi is very low thus developing a novel method for increase of the production of the polypeptide is promising. GRN polypeptides typically consist of 56-58 amino acids with six disulfide bonds and four beta-sheets to make the GRN polypeptide structurally compact and complex so large-scale preparation of natural polypeptide CS5931 is very difficult. Therefore, recombinant expression of CS5931 by genetic engineering is one of the effective ways to solve this problem. In the present study, we cloned a full length cDNA of CS5931 precursor by rapid amplification of cDNA ends (RACE) and obtained the amino acid sequence of CS5931. We further made the polypeptide expression with high efficiency in E. coli. The expressed protein was purified through affinity chromatography. The gene fragment coding CS5931 was successfully cloned and then linked into prokaryotic expression carrier pET28a(+) and pPICZαa, respectively. Two expression vectors pET28a(+)-CS5931 and pPICZαa-CS5931 were transformed into E. coli and Pichia pastoris, respectively. Then recombinant polypeptide CS5931 were successfully induced with high expression . To investigate the expression and anticancer effect of polypeptide CS5931 in different vectors and strains, we purified the expressed product developed suitable methods for CS5931 great expression while maintaining its potent anticancer activity and identified the role of CS5931 target proteins. Moreover, the purified polypeptide native CS5931 and recombinant CS5931 equally display great antitumor activity against HT29 cells. Our studies indicate that we have successfully cloned, expressed and purified Ciona savignyi polypeptide CS5931 and demonstrated its potent antitumor activity against colorectal cancer cells. Therefore, CS5931 may be developed as a novel anticancer agent in the treatment of colorectal cancer clinically. Citation Format: Yangyang Zhang, Honglin Lai, Weixiao An, Shousong Cao, Xiukun Lin. Cloning, expression and purification of Cionasavignyi polypeptide CS5931 and its antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1508.