Journal of Microbiology and Biotechnology

  • 제18권1호
  • /
  • Pages.183-187
  • /
  • 2008
  • /
  • 1017-7825(pISSN)
  • /
  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Overproduction of Recombinant Human VEGF (Vascular Endothelial Growth Factor) in Chinese Hamster Ovary Cells

  • 발행 : 2008.01.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$ $(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.

키워드

참고문헌

  1. Bae, G. W., D.-W. Jeong, H. J. Kim, G. M. Lee, H. Park, T.-B. Choe, S. Kang, I. Y. Kim, and I.-H. Kim. 2006. High productivity of t-PA in CHO cells using hypoxia response element. J. Microbiol. Biotechnol. 16: 695-703
  2. Bhisitkul, R. B. 2006. Vascular endothelial growth factor biology: Clinical implications for ocular treatments. Br. J. Ophthalmol. 90: 1542-1547 https://doi.org/10.1136/bjo.2006.098426
  3. Byun, J., J. M. Heard, J. E. Huh, S. J. Park, E. A. Jung, J. O. Jeong, H. C. Gwon, and D. K. Kim. 2001. Efficient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector. J. Mol. Cell Cardiol. 33: 295-305 https://doi.org/10.1006/jmcc.2000.1301
  4. Carmeliet, P. 2003. Angiogenesis in health and disease. Nat. Med. 9: 653-660 https://doi.org/10.1038/nm0603-653
  5. Ciudad, C. J., G. Urlaub, and L. A. Chasin. 1988. Deletion analysis of the Chinese hamster dihydrofolate reductase gene promoter. J. Biol. Chem. 263: 16274-16282
  6. Conn, G., D. D. Soderman, M. T. Schaeffer, M. Wile, V. B. Hatcher, and K. A. Thomas. 1990. Purification of glycoprotein vascular endothelial cell mitogen from rat glioma-derived cell line. Proc. Natl. Acad. Sci. USA 87: 1323-1327
  7. Ferrara, N. 2002. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: Therapeutic implication. Semin. Oncol. 29: 10-14
  8. Ferrara, N., H.-P. Gerber, and J. LeCouter. 2003. The biology of VEGF and its receptors. Nat. Med. 9: 669-676 https://doi.org/10.1038/nm0603-669
  9. Ferrara, N. 2004. Vascular endothelial growth factor: Basic science and clinical progress. Endocrine Rev. 25: 581-611 https://doi.org/10.1210/er.2003-0027
  10. Folkman, J. 1995. Angiogenesis in cancer, vascular, rheumatoid and other diseases. Nat. Med. 1: 27-31 https://doi.org/10.1038/nm0195-27
  11. Freedman, S. B. 2002. Clinical trials of gene therapy for atherosclerotic cardiovascular disease. Curr. Opin. Lipidol. 13: 653-661 https://doi.org/10.1097/00041433-200212000-00009
  12. Grabenhorst, E., P. Schlenke, S. Pohl, M. Nimtz, and H. S. Conradt. 1999. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconjugate J. 16: 81-97 https://doi.org/10.1023/A:1026466408042
  13. Henry, T. D., B. H. Annex, G. R. McKendall, M. A. Azrin, J. J. Lopez, F. J. Giordano, P. K. Shah, J. T. Willerson, R. L. Benza, D. S. Berman, C. M. Gibson, A. Bajamonde, A. C. Rundle, E. R. McCluskey; VIVA Investigators. 2003. The VIVA trials: Vascular endothelial growth factor in ischemia for vascular angiogenesis. Circulation 107: 1359-1365 https://doi.org/10.1161/01.CIR.0000061911.47710.8A
  14. Kastrup, J., E. Jorgensen, A. Ruck, K. Tagil, D. Glogar, W. Ruzyllo, H. E. Botker, D. Dudek, V. Drvota, B. Hesse, L. Thuesen, P. Blomberg, M. Gyongyosi, C. Sylven; Euroinject One Group. 2005. Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patient with stable severe angina pectoris. J. Am. Coll. Cardiol. 45: 982-988 https://doi.org/10.1016/j.jacc.2004.12.068
  15. Keyt, B. A., L. T. Berleau, H. V. Nguyen, H. Chen, H. Heinsohn, R. Vandlen, and N. Ferrara. 1996. The carboxylterminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J. Biol. Chem. 271: 7788-7795 https://doi.org/10.1074/jbc.271.13.7788
  16. Kim, E., D. Kim, H.-Y. Hwang, J. Yoon, Y. Yoon, and K. Baek. 2004. High-level expression of recombinant human interleukin- 2 in Chinese hamster ovary cells using the expression system containing transcription terminator. J. Microbiol. Biotechnol. 14:810-815
  17. Kim, J.-M., J.-S. Kim, D.-H. Park, H. S. Kang, J. Yoon, K. Baek, and Y. Yoon. 2004. Improved recombinant gene expression in CHO cells using matrix attachment regions. J. Biotechnol. 107: 95-105 https://doi.org/10.1016/j.jbiotec.2003.09.015
  18. Kozak, M. 1984. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 4: 857-872
  19. Lee, G. Y., W. W. Jung, C. S. Kang, and I. S. Bang. 2006. Expression and characterization of human endothelial growth factor (VEGF165) in insect cells. Protein Expr. Purif. 46: 503-509 https://doi.org/10.1016/j.pep.2005.09.023
  20. Mohanraj, D., T. Olson, and S. Ramakrishnan. 1995. Expression of biologically active human vascular endothelial growth factor in yeast. Growth Factors 12: 17-27 https://doi.org/10.3109/08977199509003210
  21. Na, K. H., S. C. Kim, K. S. Seo, S. H. Lee, W. B. Kim, and K. C. Lee. 2005. Purification and characterization of recombinant human follicle stimulating hormone produced by Chinese hamster ovary cells. J. Microbiol. Biotechnol. 15: 395-402
  22. Park, J., S. Yu, J. Yoon, and K. Baek. 2005. High-level expression of recombinant human bone morphogenetic protein- 4 in Chinese hamster ovary cells. J. Microbiol. Biotechnol. 15: 1397-1401
  23. Plouet, J., F. Moro, S. Bertagnolli, N. Coldeboeuf, H. Mazarguil, S. Clamens, and F. Bayard. 1997. Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect. J. Biol. Chem. 272: 13390-13396 https://doi.org/10.1074/jbc.272.20.13390
  24. Risau, W. 1997. Mechnisms of angiogenesis. Nature 386: 671- 674 https://doi.org/10.1038/386671a0
  25. Siemeister, G., B. Schnurr, K. Mohrs, C. Schachtele, D. Marme, and G. Martiny-Baron. 1996. Expression of biologically active isoforms of the tumor angiogenesis factor VEGF in Escherichia coli. Biochem. Biophys. Res. Commun. 222: 249-255 https://doi.org/10.1006/bbrc.1996.0730
  26. Tammela, T., B. Enholm, K. Alitalo, and K. Paavonen. 2005. The biology of vascular endothelial growth factors. Cardiovasc. Res. 65: 550-563 https://doi.org/10.1016/j.cardiores.2004.12.002
  27. Yamazaki, Y. and T. Morita. 2006. Molecular and functional diversity of vascular endothelial factors. Mol. Divers. 10: 515-527 https://doi.org/10.1007/s11030-006-9027-3
  28. Yang, R., S. Bunting, A. Ko, B. A. Keyt, N. B. Modi, T. F. Zioncheck, N. Ferrara, and H. Jin. 1998. Substantially attenuated hemodynamic responses to Escherichia coli-derived vascular endothelial growth factor given by intravenous infusion compared with bolus injection. J. Pharmacol. Exp. Ther. 284: 103-110