Soluble multimer of recombinant endostatin expressed in E. coli has anti-angiogenesis activity.
The bioactivity, refolding, and multimer formation of endostatin, particularly of recombinant endostatin produced from bacteria, are proved challenging for clinical application. In order to determine the biological activity of recombinant endostatin multimer, first, we expressed endostatin in Escherichia coli and purified it with ion-exchange chromatography. The purified active protein could elicit multimer formation spontaneously, but still has comparable activity. Aim to determine the anti-angiogenic activity of multimer endostatin, by use of RP-HPLC, we then successfully separated endostatin monomer and multimer for subjecting to anti-angiogenesis assay. The results from CAM (chorioallantoic membrane) inhibition assay showed that both monomer and multimer suppressed CAM vascularization significantly. At the dosage of 0.8 microg, inhibition rates of multimeric and monomeric proteins were about 58% and 38%, respectively. Multimeric endostatin exerted a higher activity than monomeric endostatin (p < 0.05). However, when the protein dosage is less than 0.4 microg/ml, there is no significance between their inhibition rates (p > 0.05), although both of them show a high inhibition effect in contrast to control. The results from HUVEC proliferation assay also showed similar effects at dosages of 0.6 and 1.6 microg/ml, multimer exerted a higher activity on inhibition of HUVEC proliferation comparing with monomer (p < 0.05). In conclusion, our results suggest that endostatin multimer has a comparable or higher bioactivity and multimerization will not affect its bioactivity, implying that endostatin activity is insensitive to structure conformation contributed by disulfide bonds.
Wei
Dong-mei
DM
Model Animal Research Center, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, PR China.
Gao
Yan
Y
Cao
Xiang-rong
XR
Zhu
Nian-chun
NC
Liang
Jian-fu
JF
Xie
Wei-ping
WP
Zhen
Ming-ying
MY
Zhu
Min-sheng
MS
D000482:Allantois / Q000098:blood supply / Q000187:drug effects; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D002823:Chorion / Q000098:blood supply / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D043169:Endostatins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D018919:Neovascularization, Physiologic / Q000187:drug effects; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility
D020533:Angiogenesis Inhibitors; D043169:Endostatins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins