Optimal production and in vitro activity of recombinant endostatin from stably transformed Drosophila melanogaster S2 cells.
Recombinant plasmids containing a complementary deoxyribonucleic acid coding mouse endostatin were transfected and stably expressed in Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing recombinant endostatin were isolated after 4 wk of selection with hygromycin B. Recombinant endostatin expressed in the stably transformed S2 cells under the influence of the Drosophila BiP protein signal sequence was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at maximum inhibition for recombinant endostatin was approximately 1.8 microg/ml. The stably transformed S2 cells produced 18 mg recombinant endostatin/L 7 d after induction with 5 microM CdCl2. Sodium butyrate supplementation (2.5 mM) increased recombinant endostatin production by 17%. These findings demonstrate optimal production and in vitro activity of recombinant endostatin from stably transformed D. melanogaster S2 cells.
Park
J H
JH
Department of Genetic Engineering, Kyung Hee University, Suwon, South Korea.
Chang
K H
KH
Lee
J M
JM
Lee
Y H
YH
Chung
I S
IS
D020533:Angiogenesis Inhibitors / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002104:Cadmium / Q000494:pharmacology; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000302:isolation & purification; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D043169:Endostatins; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D020533:Angiogenesis Inhibitors; D002087:Butyrates; D018076:DNA, Complementary; D043169:Endostatins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002104:Cadmium; D003094:Collagen; D008668:Metallothionein