[Cloning and expression of human canstatin and its purification and bioactivity].
Total RNA was extracted from placenta umbilical tissue and the canstatin cDNA was amplified from total RNA by net-RT-PCR technique. The amplified cDNA was cloned into pSP72 and sequenced. The canstatin cDNA was cut down from pSP72C with BamH I / Nde I and ligated into the vector pET-3c. The resultant plasmid pETC was then transformed into E. coli BL21 (DE3). The canstatin gene was efficiently expressed after IPTG induction as a 25 kD band on SDS-PAGE. The expressed product constituted approximately 27.9% of the total bacterial proteins estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed, refolded and purified on the Sephadex G-75 gel filtration column to a purity of 91.4%. CAM assay showed that 10 microg purified canstatin is enough to inhibit the angiogenesis of chichen embryo microcapillary vessel.
He
Guoan
G
Key Laboratory of Genetic Engineering of Ministry of Education, Zhongshan University, Guangzhou 510275, China. heguoan@263.net
Luo
Jinxian
J
Zhang
Tianyuan
T
Hu
Zhishang
Z
Li
Ruifang
R
D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002642:Chick Embryo; D003001:Cloning, Molecular; D024141:Collagen Type IV / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*
D020533:Angiogenesis Inhibitors; C502625:COL4A2 protein, human; D024141:Collagen Type IV; D010446:Peptide Fragments