Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Expression and In Vitro Activity of Recombinant Canstatin in Stably Transformed Bombyx mori Cells

  • Lee, Ji-Hye (Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University) ;
  • Lee, Jong-Min (Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University) ;
  • Jeon, Hwang-Bo (Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University) ;
  • Shon, Bong-Hee (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA) ;
  • Yang, Jai-Myung (Department of Life Science, Sogang University) ;
  • Chung, In-Sik (Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University)
  • Published : 2009.07.31
Download PDF
⟨ Previous Next ⟩

Abstract

We describe the expression of recombinant canstatin from stably transformed Bombyx mori BmS (BmS) 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 ($ED_{50}$) for recombinant canstatin expressed in stably transformed BmS cells was approximately 0.64 ${\mu}g/ml$. A maximum production level of 11 mg/l recombinant canstatin was obtained in a T-flask culture of BmS cells after 6 days of incubation.

Keywords

References

  1. Finney, D. J. 1971. Probit Analysis, Third Ed. Cambridge University Press, Cambridge
  2. Folkman, J. 2006. Angiogenesis. Annu. Rev. Med. 57: 1-18 https://doi.org/10.1146/annurev.med.57.121304.131306
  3. He, G. A., J. X. Luo, T. Y. Zhang, Z. S. Hu, and F. Y. Wang. 2004. The C-terminal domain of canstatin suppresses in vivo tumor growth associated with proliferation of endothelial cells. Biochem. Biophys. Res. Commun. 318: 354-360 https://doi.org/10.1016/j.bbrc.2004.04.038
  4. Hou, W. H., T. Y. Wang, B. M. Yuan, Y. R. Chai, Y. L. Jia, F. Tian, J. M. Wang, and L. X. Xue. 2004. Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell proliferation. Acta Biochim. Biophys. Sin. (Shanghai) 36: 845-850 https://doi.org/10.1093/abbs/36.12.845
  5. Kamphaus, G. D., P. C. Colorado, D. J. Panka, H. Hopfer, R. Ramchandran, A. Torre, et al. 2000. Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth. J. Biol. Chem. 275: 1209-1215 https://doi.org/10.1074/jbc.275.2.1209
  6. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of head of bacteriophage. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  7. Lee, J. M., J. Hwang-Bo, B. H. Sohn, and I. S. Chung. 2007. Functional expression of recombinant canstatin in stably transformed Drosophila melanogaster S2 cells. Protein Expr. Purif. 52: 258-264 https://doi.org/10.1016/j.pep.2006.11.016
  8. Maeshim, Y., P. C. Colorado, A. Torre, K. A. Holthaus, J. A. Grunkemeyer, M. B. Ericksen, H. Hopfer, Y. Xiao, I. E. Stillman, and R. Kalluri. 2000. Distinct antitumor properties of a type IV collagen domain derived from basement membrane. J. Biol. Chem. 275: 21340-21348 https://doi.org/10.1074/jbc.M001956200
  9. Magnon, C., A. Galaup, B. Mullan, V. Rouffiac, J. M. Bidart, F. Griscelli, P. Opolon, and M. Perricaudet. 2005. Canstatin acts on endothelial and tumor cells via mitochondrial damage initiated through interaction with $\alpha_v\beta_3$ and $\alpha_v\beta_5$ integrins. Cancer Res. 65: 4353-4361 https://doi.org/10.1158/0008-5472.CAN-04-3536
  10. O'eilly, M. S., T. Boehm, Y. Shing, N. Fukai, G. Vasios, W. S. Lane, et al. 1997. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277-285 https://doi.org/10.1016/S0092-8674(00)81848-6
  11. Panka, D. J. and J. W. Mier. 2003. Canstatin inhibits Akt activation and induces Fas-dependent apoptosis in endothelial cells. J. Biol. Chem. 278: 37632-37636 https://doi.org/10.1074/jbc.M307339200
  12. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbor, New York