Expression of soluble, biologically active recombinant human endostatin in Escherichia coli.
Endostatin, a 20kDa C-terminal fragment of collagen XVIII, is a potent anti-angiogenic protein and inhibitor of tumor growth. Recombinant endostatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in E. coli by employing both co-expression of the molecular chaperones and lower temperature fermentation. Two groups of chaperones Trigger factor and GroEL-GroES (GroEL/ES), DnaK-DnaJ-GrpE and GroEL/ES, were co-expressed, respectively, with rhEndostatin at different temperatures (37, 25, and 16 degrees C). It revealed that low temperature or molecular chaperones alone could enhance the production of active rhEndostatin; meanwhile, combinational employment of low temperature cultivation (16 degrees C) together with co-expression of DnaK-DnaJ-GrpE and GroEL/ES was more effective to prevent aggregation of rhEndostatin. The production of soluble rhEndostatin was about 36 mg/L, and at least 16 mg of rhEndostatin was purified from 1L flask culture. The purified rhEndostatin specifically inhibited the proliferation of endothelial cell-bovine capillary endothelial cell in a dose-dependent manner, and it showed potent anti-angiogenic capability on the chorioallantoic membrane of chick embryo in vivo. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.
Xu
Han-Mei
HM
The State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing 210093, PR China.
Zhang
Guo-Yuan
GY
Ji
Xiao-Dan
XD
Cao
Lin
L
Shu
Luan
L
Hua
Zi-Chun
ZC
D016475:3T3 Cells; D020533:Angiogenesis Inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D000818:Animals; D001681:Biological Assay; D002196:Capillaries / Q000166:cytology / Q000187:drug effects; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D043169:Endostatins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D013696:Temperature
D020533:Angiogenesis Inhibitors; D043169:Endostatins; D018832:Molecular Chaperones; D011994:Recombinant Proteins