|
|
|
| Expression of Fusion Protein ES-Kringle5 and Its Purification and Biological Analysis |
| CHEN Yue, FU Zhong-ping, LI Jing-rong, YIN Xiao-jin |
| Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing 210042, China |
|
|
|
Abstract Objective：To express recombinant ES-Kringle5 fusion protein in prokaryotic cells, purify and identify the bioactivity of expressed product. Method：The nucleotide sequence encoding the 27-amino-acid peptide corresponding to the NH2-terminal domain of endostatin and Kringle 5 via a peptide linker were synthesized and inserted to pMD18-T, then the sequence cloned into vector pET25b. And this recombinant plasmid was transformed to E.coli BL21(DE3) for expression by lactose induction. The expression product was purified by Ni-NTA. The abilities of ES-Kringle 5 to inhibit the proliferation of HUVECs was used for its biological activity assay. Results: The ES-Kringle5 coding sequence was correctly cloned into pET-25b vector. Use lactose and lower the induction temperature can increase the expression level and soluble protein expression. The recombinant protein reached a purity of more than 95% after purification. Bioactivity assay result shows that ES-Kringle5 could inhibit the proliferation of HUVECs. Conclusion：The recombinant ES-Kringle5 fusion protein was successfully expressed in E.coli with high biological activity, which lay the material foundation for its pharmacology study in vivo.
|
|
Received: 13 March 2014
Published: 25 May 2014
|
|
|
|
|
|
[1] Folkman J. Tumor angiogenesis : therapeutic implications. N Engl J Med,1971,285 (21) : 1182 - 1186.
[2] Folkman J. Antiangiogenesis in cancer therapy endostatin and its mechanisms of action. Experi mental Cell Research,2006,312 : 594-607.
[3] Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev,2004,25(4) : 581-611.
[4] O'Reilly M S,Hohngren L,Shing Y,et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastase by a Lewis lung carcinoma. Cell,1994,79(2) : 315-328.
[5] Sottile J. Regulation of angiogenesis by extracellular matrix. Biochinl Biophys Acta,2004,1654(1) : 13-22.
[6] O'Reilly M S,Boehm T. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell,1997,8 : 277-285.
[7] Yumi Y,Mohanraj D,Arjan W G,et al. Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth. Cancer Res,2000,60 : 2190-2196.
[8] Frank A S,Anthony C,Smith R,et al. Statin-AE: A novel angiostatin-endostatin fusion protein with enhanced antiangiogenic and antitumor activity. Angiogenesis,2001,4 : 263-268.
[9] Frank A S,Smith R,Pathak A,et al. Combination angiostatin and endostatin gene transfer induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid tumors in mice. Molecular Therapy,2001,3(2) : 186-196.
[10] Chen J S,Hwang J C,Chang L C,et al. Attributes of antiangiogenic factor plasminogen kringle 5 in glomerulonephritis. Arch Pathol Lab Med,2010,134(12) : 1804-1812.
[11] Li C,Li L,Cheng R,et al. Acidic/neutral amino acid residues substitution in NH2 terminal of plasminogen Kringle 5 exerts enhanced effects on corneal neovascularization. Cornea,2013,32(5) : 680-688.
[12] Robert M T,Ronit S F,Amy E B,et al. A 27-Amino-Acid Synthetic Peptide Corresponding to the NH2-Terminal Zinc-Binding Domain of Endostatin Is Responsible for Its Antitumor Activity. Cancer Res,2005,65(9) : 3656-63.
[13] Folkman J. Antiangiogenesis in cancer therapy—endostatin and its mechanisms of action. Exp Cell Res,2006,312 (5) : 594-607.
[14] Kashi J,Lee T Y,Robert M,et al. Two endogeneous antiangiogenic inhibitors, endostatin and angiostatin, demonstrate biphasic curves in their antitumor profiles. Dose-Response,2011,9 : 369-376.
[15] 王琪,李嘉姝,程莉莉. 抗血管生成与肿瘤治疗. 检验医学与临床,2012,9(1) : 56-58. Wang Q, Li J S, Cheng L L. Antiangiogenesis and tumor therapeutics. Labortory Medicine and Clinic,2012,9(1) : 56-58.
[16] Zhu D G,Su W,Jin G S,et al. Glioma stem cells targeted by oncolytic virus carrying endostatin-angiostatin fusion gene and the expression of its exogenous gene in vitro. Brain Research,2011,59-69.
[17] Zhang W,Fulci G L,Wakimoto H,et al. Combination of oncolytic herpes simplex viruses armed with angiostatin and IL-12 enhances antitumor efficacy in human glioblastoma models. Neoplasia,2013,15 : 591-599.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
