title: Expression and In vitro activity of recombinant canstatin in stably transformed bombyx mori cells.
abstract: 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.
authors: Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Suwon, Korea.
authors: Ji Hye
authors: JH
authors: Lee
authors: Jong Min
authors: JM
authors: Lee
authors: Hwang-Bo
authors: HB
authors: Jeon
authors: Bong Hee
authors: BH
authors: Shon
authors: Jai Myung
authors: JM
authors: Yang
authors: In Sik
authors: IS
authors: Chung
mesh_terms: 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
chemical_list: D020533:Angiogenesis Inhibitors; C502625:COL4A2 protein, human; D024141:Collagen Type IV; D010446:Peptide Fragments; D011994:Recombinant Proteins