Expression and In vitro activity of recombinant canstatin in stably transformed bombyx mori cells.
We describe the expression of recombinant canstatin from stably transformed Bombyx mori Bm5 (Bm5) cells. Recombinant canstatin was secreted into a culture medium with a molecular mass of approximately 29 kDa. Densitometric scanning showed that the secreted canstatin accounted for approximately 91% of the total canstatin production. Recombinant canstatin was also purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. The identity of the purified protein was confirmed as human canstatin by nano-LC-MS/MS analysis. Purified recombinant canstatin inhibited human endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant canstatin expressed in stably transformed Bm5 cells was approximately 0.64 mg/ml. A maximum production level of 11 mg/l recombinant canstatin was obtained in a T-flask culture of Bm5 cells after 6 days of incubation.
Lee
Ji Hye
JH
Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Suwon, Korea.
Lee
Jong Min
JM
Jeon
Hwang-Bo
HB
Shon
Bong Hee
BH
Yang
Jai Myung
JM
Chung
In Sik
IS
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000008:administration & dosage / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D012831:Bombyx; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D024141:Collagen Type IV / Q000008:administration & dosage / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology; D005590:Fractional Precipitation; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000008:administration & dosage / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
D020533:Angiogenesis Inhibitors; C502625:COL4A2 protein, human; D024141:Collagen Type IV; D010446:Peptide Fragments; D011994:Recombinant Proteins