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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Amino Acid Sequence
-
Angiogenesis Inhibitors / administration & dosage
-
Angiogenesis Inhibitors / isolation & purification
-
Angiogenesis Inhibitors / metabolism
-
Animals
-
Bombyx
-
Cell Line
-
Cell Proliferation / drug effects
-
Collagen Type IV / administration & dosage
-
Collagen Type IV / isolation & purification
-
Collagen Type IV / metabolism*
-
Dose-Response Relationship, Drug
-
Endothelial Cells / drug effects
-
Endothelial Cells / physiology
-
Fractional Precipitation
-
Humans
-
Molecular Sequence Data
-
Peptide Fragments / administration & dosage
-
Peptide Fragments / isolation & purification
-
Peptide Fragments / metabolism*
-
Recombinant Proteins / administration & dosage
-
Recombinant Proteins / isolation & purification
-
Recombinant Proteins / metabolism
-
Time Factors
Substances
-
Angiogenesis Inhibitors
-
COL4A2 protein, human
-
Collagen Type IV
-
Peptide Fragments
-
Recombinant Proteins