|
|
|
| Prokaryotic Expression, Purification and Antitumor Activity Identification of Tumor Targeting iRGD-CDD Fusion Protein |
| WANG Qi-fan, XUE Ying, FENG Xin-wei, ZHANG Ge |
| Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China |
|
|
|
Abstract Objective: To express tumor targeting GP-CDD-iRGD fusion protein with specific fibroblast activation protein (FAPα) restricting sites in prokaryotic cells,purify the expressed product and determine its toxicity to FAPα positive and negative tumor cell lines, evaluate the feasibility to remove the fusion tag in vivo. Methods: Design and synthesis the gene of GP-CDD-iRGD, insert it into pGEX-4T3 vector. The recombinant plasmid was transformed into E. coli BL21 cells for expression under induction of IPTG. The expressed recombinant fusion protein was analyzed for expression level and form by SDS-PAGE, then purified by GST affinity tag protein purification kit, evaluate the anti-tumor activity and FAPα targeted cleavage effect through in vitro toxicity tests. Results: Restriction analysis and sequencing proved that recombinant plasmid pGEX-GP-CDD-iRGD was constructed correctly. Recombinant GP-CDD-iRGD fusion protein, with a relative molecular mass of about 36000, was solublely expressed and reached a purity of about 90% after purification.ED50 of the protein is about 18.5μmol/L. Conclusion: It showed significant toxicity to FAPα-positive 4T1 cells while FAPα-negative cell lines with no effect which established a foundation of further reasearch of its antitumor activity in vivo.
|
|
Received: 18 September 2014
Published: 25 December 2014
|
|
|
|
|
|
[1] Svensen N, Walton J G, Bradley M. Peptides for cell-selective drug delivery. Trends Pharmacol Sci, 2012,33(4):186-192.
[2] 闫国婷,胡军平, 张宇婷等, 抗肿瘤靶向药物最新研究进展. 煤炭与化工, 2013,5:59-60,67. Yan G T,et al. The lastest research process of antitumor targeted drugs. Coal and Chemical Industry,2013,5:59-60,67.
[3] Sugahara K N, et al. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science, 2010,328(5981):1031-1035.
[4] Jan Y, Matter M, Pai J T,et al. A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors. Cell, 2004,116(5):751-762.
[5] Chen R, et al. Application of a proapoptotic peptide to intratumorally spreading cancer therapy. Cancer Res, 2013,73(4):1352-1361.
[6] Milner J M, et al. Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis. Arthritis Res Ther, 2006,8(1):23.
[7] LeBeau A M, et al. Targeting the cancer stroma with a fibroblast activation protein-activated promelittin protoxin. Mol Cancer Ther, 2009,8(5):1378-1386.
[8] 马伟峰,杨飞华,赵海山,等. FAPα酶激活式靶向抗肿瘤前药:Z-GP-Dox对斑马鱼的毒性评价. 中国生物工程杂志, 2012,7:37-42. Ma W F,Yang F H,Zhao H S,et al. Toxicity Evaluation of a FAPα-activated targeting anticancer prodrug Z-GP-Dox in zebrafish. China Biotechnology,2012,7:37-42.
[9] Radha G V, Rani T S, Sarvani B. A review on proniosomal drug delivery system for targeted drug action. J Basic Clin Pharm, 2013,4(2):42-48.
[10] Pasquetto M V, et al. Targeted drug delivery using immunoconjugates: principles and applications. J Immunother, 2011,34(9):611-628.
[11] 韩笑, 李娜,杜培革. 抗肿瘤多肽研究进展. 中国生物工程杂志, 2013,6:93-98. Han X, Li N,Du P G,et al. Recent development of antitumor peptides. China Biotechnology, 2013,6:93-98.
[12] 曾名嘉, 张冬梅,陈钧辉. 抗肿瘤多肽研究进展. 中国生化药物杂志, 2007,2:139-141. Zeng M J,Zhang D M,Chen J H.Research advances of antitumor peptides.Chinese Journal of Biochemical Pharmaceutics,2007,2:139-141.
[13] Wayne A S, et al. Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial. Clin Cancer Res, 2010,16(6): 1894-1903.
[14] Woo J H, et al. GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials. Protein Expr Purif, 2008,58(1):1-11.
[15] Tong M, et al. Prevention of postoperative recurrence of human bladder carcinoma by intravesical instillation of immunotoxin, a clinical study. Zhonghua Yi Xue Za Zhi, 2003,83(3):201-203.
[16] 杨永晶, 徐寒梅,胡加亮. 以整合素为靶点的抗肿瘤药物研究进展. 药物生物技术, 2012,3:256-260. Yang Y J,Xu H M, Hu J L. Drug development in antitumor therapy by targeting of integrin. Pharmaceutical Biotechnology,2012,3:256-260.
[17] 曾凡伟,曹畅,吕莎,等. 靶向Neuropilin-1的抗肿瘤研究进展. 中国生化药物杂志, 2012,5(6):679-682. Zen F W,Cao C,Lv S,et al. The antitumor research progress in targeting neuropilin-1.Chinese Journal of Biochemical Pharmaceutics,2012,5:679-682.
[18] Vasina J A, Baneyx F. Expression of aggregation-prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expr Purif, 1997,9(2):211-218.
[19] Weickert M J, et al. Optimization of heterologous protein production in Escherichia coli. Curr Opin Biotechnol, 1996. 7(5):494-499.
[20] 肖斌,杨珺,朱永红,等. 靶向性抗肿瘤融合蛋白RGD-hIL-24的构建、表达和体外活性研究. 中华微生物学和免疫学杂志, 2006,12: 1059-1063. Xiao B,Yang J,Zhu Y H,et al.The construction and expression of tumor targeting protein RGD-H-L24 and the study of its in vitro antitumor acitivity. Chin J Microbiol Immunal,2006,12:1059-1063.
[21] 卜鹏莉,王东梅,陈虹,等. 靶向性抗肿瘤融合蛋白EGF-E4orf4的表达及其细胞毒性的初步分析. 癌症, 2005,1:33-39. Bu P L,Wang D M,Chen H,et al. Expression of epidermal growthfactor-adenovirusE4orf4 fusion protein and its cytotoxicity.Chinese Journal of Cancer, 2005,1:33-39.
[22] Kaspar A A, Reichert J M. Future directions for peptide therapeutics development. Drug Discov Today, 2013,18(17-18): 807-817.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
