title: Expression of soluble, biologically active recombinant human tumstatin in Escherichia coli.
abstract: Tumstatin, a 28-kDa C-terminal fragment of collagen IV, is a potent anti-angiogenic protein and inhibitor of tumour growth. Recombinant tumstatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human tumstatin in E. coli by the coding region of tumstatin being linked to the 3'-end of the maltose-binding protein (MBP) gene. The fusion protein was expressed as the soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tumstatin was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 20% of the total soluble protein in E. coli, allowing approximately 8.6 mg of highly purified protein to be obtained per litre of bacterial culture. The purified tumstatin specifically inhibited the proliferation of endothelial cells in a dose-dependent manner. Annexin V-FITC apoptotic assay showed that recombinant tumstatin induced significant increase of apoptotic endothelial cells after 20 h of exposure to 20 microg/ml tumstatin, and when tumstatin was incubated on the chicken embryo, chorioallantoic membrane at doses of 1-15 microg, there was a dramatic decrease in the microvasculature allantoids of chicken embryos neovascular vessel test in vivo demonstrated that tumstatin treatment at doses of 1-15 microg gives rise to dramatically decrease the number of neovascular vessel. Our study provides a feasible and convenient approach to produce soluble and biologically active tumstatin.
authors.affiliation: Department of Laboratory Medicine, The Affiliated Anhui Provincial Hospital of Anhui Medical University, Hefei 23001, China. luoyiqin2004@hotmail.com
authors.forename: Yi-Qin
authors.initials: YQ
authors.lastname: Luo
authors.forename: Liang-Hua
authors.initials: LH
authors.lastname: Wang
authors.forename: Qui
authors.initials: Q
authors.lastname: Yi
authors.forename: Bing-Hua
authors.initials: BH
authors.lastname: Jiao
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D024141:Collagen Type IV / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D058619:Maltose-Binding Proteins; D018919:Neovascularization, Physiologic / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D012995:Solubility
chemical_list: D001324:Autoantigens; D002352:Carrier Proteins; D024141:Collagen Type IV; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; C057112:type IV collagen alpha3 chain