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 29 kDa was secreted into the culture medium. Recombinant canstatin was purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation. Purified recombinant canstatin inhibited human umbilical vein endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED₅₀) for recombinant canstatin expressed in stably transformed Drosophila S2 cells was approximately 0.37 μg/ml. A maximum production level of 76 mg/l of recombinant canstatin was obtained in a T-flask culture of Drosophila S2 cells 6 days after induction with 0.5 mM CuSO₄.