Approaches to efficient production of recombinant angiogenesis inhibitor rhVEGI-192 and characterization of its structure and antiangiogenic function.
Methods to prepare pure, bioactive recombinant human vascular endothelial growth inhibitor (rhVEGI), a potent inhibitor of angiogenesis potentially applicable in antiangiogenic cancer therapy, are in urgent demand for preclinical investigation as well as future clinical trials of the protein. Here, we report expression and purification of rhVEGI-192, a recombinant VEGI isoform, comparatively using host strains BL21 (DE3) pLysS and Origami B (DE3) with IPTG-induction and autoinduction techniques. Our study identified that a combined use of Origami B (DE3) strain and autoinduction expression system gave rise to a high yield of purified rhVEGI-192 at 105.38 mg/L culture by immobilized-metal affinity chromatography on Ni-NTA column. The antiangiogenic activity was effectively restored after the insoluble fractions being dissolved in 8M urea and subsequently subjected to a gradient-dialysis refolding process. Functional tests demonstrated that the purified rhVEGI-192 potently inhibited endothelial growth, induced endothelial apoptosis and suppressed neovascularization in chicken chorioallantoic membrane, indicating that the developed method allows preparation of rhVEGI-192 with high yield, solubility, and bioactivity. Most importantly, our study also demonstrates that VEGI-192 is capable of forming polymeric structure, which is possibly required for its antiangiogenic activity.
Chen
Xu
X
Department of Clinical Medicine, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China.
Wu
Jueheng
J
Liu
Hanning
H
He
Zhenjian
Z
Gu
Minghui
M
Wang
Ning
N
Ma
Jianda
J
Hu
Jieping
J
Xia
Lei
L
He
Haipeng
H
Yuan
Jie
J
Li
Jun
J
Li
Luyuan
L
Li
Mengfeng
M
Zhu
Xun
X
D020533:Angiogenesis Inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D018929:Cell Culture Techniques; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000187:drug effects; D002846:Chromatography, Affinity; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D053380:Tumor Necrosis Factor Ligand Superfamily Member 15 / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D011994:Recombinant Proteins; C117026:TNFSF15 protein, human; D053380:Tumor Necrosis Factor Ligand Superfamily Member 15