Functional expression of recombinant canstatin in stably transformed Drosophila melanogaster S2 cells.
We describe the expression and in vitro activity of recombinant canstatin from stably transformed Drosophila melanogaster S2 cells. Southern blot analysis indicated that transformed S2 cells contained multiple copies of the canstatin gene in the genome. Recombinant canstatin with a molecular weight of 29kDa was secreted into the culture medium. Recombinant canstatin was purified to homogeneity using a simple one-step Ni(2+) affinity fractionation. Purified recombinant canstatin inhibited human umbilical vein endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED(50)) for recombinant canstatin expressed in stably transformed Drosophila S2 cells was approximately 0.37mug/ml. A maximum production level of 76mg/l of recombinant canstatin was obtained in a T-flask culture of Drosophila S2 cells 6 days after induction with 0.5mM CuSO(4).
Lee
Jong Min
JM
Department of Genetic Engineering and Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701, Republic of Korea.
Jeon
Hwang-Bo
HB
Sohn
Bong Hee
BH
Chung
In Sik
IS
D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology*; D042783:Endothelial Cells / Q000166:cytology; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors
C502625:COL4A2 protein, human; D024141:Collagen Type IV; D010446:Peptide Fragments; D011994:Recombinant Proteins