[Expression of human tumstatin in Pichia pastoris and its bioactivity].
Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.
Gu
Qu-Liang
QL
Key Laboratory of Gene Engineering of Ministry of Education, Sun Yat-Sen University, Guangzhou 510275, China.
Zhang
Tian-Yuan
TY
Luo
Jin-Xian
JX
Gan
Jing-Jing
JJ
Xiao
Fan
F
D020533:Angiogenesis Inhibitors / Q000378:metabolism*; D001324:Autoantigens / Q000235:genetics* / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D018274:Electroporation; D042783:Endothelial Cells / Q000166:cytology; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014470:Umbilical Cord / Q000166:cytology
D020533:Angiogenesis Inhibitors; D001324:Autoantigens; D024141:Collagen Type IV; D018076:DNA, Complementary; D011994:Recombinant Proteins; C057112:type IV collagen alpha3 chain