Expression, purification, and bioactivity of human tumstatin from Escherichia coli.
Tumstatin is a M(r) 28,000 C-terminal NC1 fragment of type alpha3 (IV) collagen that inhibits pathological angiogenesis and suppresses proliferation of endothelial cells and growth of tumors. We report here high cytoplasmic expression of recombinant human tumstatin in Escherichia coli and its purification, in vitro refolding, and inhibitory activity analysis. Human tumstatin was expressed in the bacterial cytoplasm as an insoluble N-terminal polyhistidine tagged protein, which accounted for more than 30% of total bacterial protein in BL21 (DE3) cells. After extraction and solubilization in guanidine-HCl, recombinant protein was purified to homogeneity using a simple one-step Ni(2+)-chelate affinity chromatography and then refolded by dialysis against acidic pH buffers with gradually decreasing concentrations of denaturant. The renatured recombinant tumstatin could specifically inhibit endothelial cell proliferation in a dose-dependent manner, and suppress bFGF-induced angiogenesis in chick embryo chorioallantoic membrane and tumor growth in mouse B16 melanoma xenograft models.
Gu
Quliang
Q
The Key Laboratory of Gene Engineering of Ministry of Education, Sun Yat-Sen University, Guangzhou, China.
Zhang
Tianyuan
T
Luo
Jinxian
J
Wang
Fangyu
F
D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000187:drug effects / Q000378:metabolism; D002846:Chromatography, Affinity; D024141:Collagen Type IV / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D004926:Escherichia coli / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008545:Melanoma / Q000188:drug therapy / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D014183:Transplantation, Heterologous
D001324:Autoantigens; D024141:Collagen Type IV; D011994:Recombinant Proteins; C057112:type IV collagen alpha3 chain; D016222:Fibroblast Growth Factor 2