Expression and purification of human endostatin from Hansenula polymorpha A16.
Endostatin (EDN), an endogenous angiogenesis inhibitor of 20 kDa, was originally isolated from the supernatant of culture murine hemangioendothelioma cell line. Interest in EDN arises from its therapeutic potential as anti-tumor and anti-angiogenesis agents. However, it is difficult to obtain sufficient quantities of native EDN from its natural resources. We report here the construction of a pMIRH-type vector pLRG29 by introducing the 25s rDNA of Hansenua wingei and the Km(R) gene into the YIp vector pSML12 and the EDN expression vector pLRG-EDN by cloning the human EDN cDNA into pLRG29. Human EDN was expressed in Hansenula polymorpha (H.p.) A16 (pLRG-EDN) as a secreted soluble protein. The yield of the secreted EDN was 65 mg/L in shake flask. The secreted EDN was purified to a purity of 98 % by the use of SP Sepharose FF ion-exchange chromatography and Sepharose-heparin Hi Trap affinity chromatography. The MTT and chicken embryo chorioallantoic membrane assay demonstrated that the human EDN produced from H. polymorpha inhibited in vitro the proliferation of human umbilical vein endothelial cells and in vivo the neovascularization induced by bFGF.
Wu
Jiangxue
J
The Key Laboratory of Genetic Engineering of Ministry of Education, Department of Biochemistry, Sun Yat-Sen University, Guangzhou, PR China.
Fu
Wuzhao
W
Luo
Jinxian
J
Zhang
Tianyuan
T
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D043169:Endostatins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D014170:Transformation, Genetic / Q000235:genetics; D014471:Umbilical Veins / Q000166:cytology
D043169:Endostatins; D011994:Recombinant Proteins