Expression and Purification of Soluble Recombinant Human Endostatin in Escherichia coli

다국어 초록 (Multilingual Abstract)

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography. As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.

참고문헌 (Reference)

1 Ding, Y. H, "Zinc-dependent dimmers observed in crystals of human endostatin" 95 : 10443-10448, 1998

2 Boehm, T, "Zinc-binding of endostatin is essential for its antiangiogenic activity" 252 : 190-194, 1998

3 Sasaki, T, "Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin" 17 : 4249-4256, 1998

4 You, W. K, "Purification and characterization of recombinant murine endostatin in Escherichia coli" 31 : 197-202, 1999

5 Davis, G. D, "New fusion protein systems designed to give soluble expression in Escherichia coli" 65 : 382-388, 1999

6 Violand, B. N, "Method of producing mouse and human endostatin"

7 Sudhakar, A, "Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by αVβ3 and α5β1 integrins" 100 : 4766-4771, 2003

8 Chura-Chambi, R. M, "High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells" 35 : 11-16, 2004

9 Sun, Z, "Expression, purification, and characterization of human urodilatin in Escherichia coli" 55 : 312-318, 2007

10 Xu, H. M, "Expression of soluble, biologically active recombinant human endostatin in Escherichia coli" 41 : 252-258, 2005

11 Wohlkonig, A, "Expression and purification in high yield of a functionally active recombinant human Type I inositol (1,4,5)P3 5-phosphatase" 55 : 69-74, 2007

12 Yan, B, "Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737" 236 : 129-136, 1737

13 Dhanabal, M, "Endostatin: Yeast production. Mutants and antitumor effect in renal cell carcinoma" 59 : 189-197, 1999

14 O’Reilly, M. S, "Endostatin: An endogenous inhibitor of angiogenesis and tumor growth" 88 : 277-285, 1997

15 Bessette, P. H, "Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm" 96 : 13703-13708, 1999

16 Hohenester, E, "Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution" 17 : 1656-1664, 1998

17 Niiranen, L, "Compara­tive expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli" 52 : 210-218, 2007

18 Dhanabal, M, "Cloning, expression, and in vitro activity of human endostatin" 258 : 345-352, 1999

19 Boehm, T, "Antiangio­genic therapy of experimental cancer does not induce acquired drug resistance" 390 : 404-407, 1997

20 Miosge, N, "Angiogenesis inhibitor endostatin is a distinct component of elastic fibers in vessel walls" 13 : 1743-1750, 1999

21 Bradford,M.M, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding" 72 : 248-254, 1976