Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli.
Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni-NTA (Ni(2+)-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.
Medical Biology Research Center, Kermanshah University of Medical Sciences, P.O. Box 6714869914, Sorkheh lizheh, Kermanshah, Iran.
Kheirollah
K
Yari
Saboor
S
Afzali
Hadi
H
Mozafari
Kamran
K
Mansouri
Ali
A
Mostafaie
D020533:Angiogenesis Inhibitors / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D000818:Animals; D002196:Capillaries / Q000187:drug effects / Q000473:pathology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004354:Drug Screening Assays, Antitumor; D043169:Endostatins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D004926:Escherichia coli; D005285:Fermentation; D015870:Gene Expression; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D051379:Mice; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*
D020533:Angiogenesis Inhibitors; D043169:Endostatins; D011994:Recombinant Proteins