Journal of Microbiology and Biotechnology

  • 제21권12호
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  • Pages.1299-1305
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  • 2011
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Combined TGE-SGE Expression of Novel PAI-1-Resistant t-PA in CHO DG44 Cells Using Orbitally Shaking Disposable Bioreactors

  • 투고 : 2011.06.01
  • 심사 : 2011.07.25
  • 발행 : 2011.12.28
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초록

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncated-mutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncated-mutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

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참고문헌

  1. American Heart Association. 2008. Heart Disease and Stroke Statistics. Accessible at http://www.americanheart.org/downloadable/ heart/1200082005246HS_Stats%202008.final.pdf.
  2. Backliwal, G., M. Hildinger, S. Chenuet, S. Wulhfard, J. M. De, and F. M. Wurm. 2008. Rational vector design and multipathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res. 36: e96. https://doi.org/10.1093/nar/gkn423
  3. Baldi, L., D. L. Hacker, M. Adam, and F. M. Wurm. 2007. Recombinant protein production by large-scale transient gene expression in mammalian cells: State of the art and future perspectives. Biotechnol. Lett. 29: 677-684. https://doi.org/10.1007/s10529-006-9297-y
  4. Baruah, D. B., R. N. Dash, M. R. Chaudhari, and S. S. Kadam. 2006. Plasminogen activators: A comparison. Vascul. Pharmacol. 44: 1-9. https://doi.org/10.1016/j.vph.2005.09.003
  5. Benavides Damm, T. and F. M. Wurm. 2010. Generation of the stable cell line for the production of theraputic proteins using the Piggy Back transposon for transgene delivery. Master Thesis Project.
  6. Benchenane, K., J. P. Lopez-Atalaya, M. Fernandez-Monreal, O. Touzani, and D. Vivien. 2004. Equivocal roles of tissue-type plasminogen activator in stroke-induced injury. Trends Neurosci. 27: 155-160. https://doi.org/10.1016/j.tins.2003.12.011
  7. Burck, P. J., D. H. Berg, M. W. Warrick, D. T. Berg, J. D. Walls, S. R. Jaskunas, et al. 1990. Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain. J. Biol. Chem. 265: 5170-5177.
  8. Davami, F., S. Sardari, A. Majidzadeh, M. Hemayatkar, F. Barkhordari, S. Enayati, et al. 2011. A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments. BMB Rep. 44: 34-39. https://doi.org/10.5483/BMBRep.2011.44.1.34
  9. Davami, F., S. Sardari, A. Majidzadeh, M. Hemayatkar, F. Barkhrdari, M. Omidi, et al. 2010. Expression of a novel chimeric truncated t-PA in CHO cells based on in silico experiments. J. Biomed. Biotechnol. 2010: 108159.
  10. Erickson, L. A., M. H. Ginsberg, and D. J. Loskutoff. 1984. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets. J. Clin. Invest. 74: 1465-1472. https://doi.org/10.1172/JCI111559
  11. Hemayatkar, M., F. Mahboudi, A. Majidzadeh, F. Davami, B. Vaziri, F. Barkhordari, et al. 2010. Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae. Biotechnol. J. 5: 1198-1206. https://doi.org/10.1002/biot.201000233
  12. Krishnamurti, C. and B. M. Alving. 1992. Plasminogen activator inhibitor type 1: Biochemistry and evidence for modulation of fibrinolysis in vivo. Semin. Thromb. Hemost. 18: 67-80. https://doi.org/10.1055/s-2007-1002412
  13. Krishnamurti, C., B. Keyt, P. Maglasang, and B. M. Alving. 1996. PAI-1-resistant t-PA: Low doses prevent fibrin deposition in rabbits with increased PAI-1 activity. Blood 87: 14-19.
  14. Krishnamurti, C., G. D. Young, C. F. Barr, C. A. Colleton, and B. M. Alving. 1991. Enhancement of tissue plasminogen activatorinduced fibrinolysis by activated protein C in endotoxin-treated rabbits. J. Lab. Clin. Med. 118: 523-530.
  15. Li, X. K., H. R. Lijnen, L. Nelles, H. B. Van, J. M. Stassen, and D. Collen. 1992. Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1. Blood 79: 417-429.
  16. Majidzadeh, A., V. Khalaj, D. Fatemeh, H. Mahdi, B. Farzaneh, A. Ahmad, and F. Mahboudi. 2010. Cloning and expression of functional full-length human tissue plasminogen activator in Pichia pastoris. Appl. Biochem. Biotechnol. 162: 2037-2048. https://doi.org/10.1007/s12010-010-8979-z
  17. Mitsuyama, K., M. Sata, and S. Rose-John. 2006. Interleukin-6 trans-signaling in inflammatory bowel disease. Cytokine Growth Factor Rev. 17: 451-461. https://doi.org/10.1016/j.cytogfr.2006.09.003
  18. Nordt, T. K. and C. Bode. 2003. Thrombolysis: Newer thrombolytic agents and their role in clinical medicine. Heart 89: 1358-1362. https://doi.org/10.1136/heart.89.11.1358
  19. Novick, D. and M. Rubinstein. 2007. The tale of soluble receptors and binding proteins: From bench to bedside. Cytokine Growth Factor Rev. 18: 525-533. https://doi.org/10.1016/j.cytogfr.2007.06.024
  20. Paoni, N. F., B. A. Keyt, C. J. Refino, A. M. Chow, H. V. Nguyen, L. T. Berleau, et al. 1993. A slow clearing, fibrinspecific, PAI-1 resistant variant of t-PA (T103N, KHRR 296-299 AAAA). Thromb. Haemost. 70: 307-312.
  21. Pennica, D., W. E. Holmes, W. J. Kohr, R. N. Harkins, G. A. Vehar, C. A. Ward, et al. 1983. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 301: 214-221. https://doi.org/10.1038/301214a0
  22. Pham, P. L., A. Kamen, and Y. Durocher. 2006. Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol. Biotechnol. 34: 225-237. https://doi.org/10.1385/MB:34:2:225
  23. Ranby, M., N. Bergsdorf, G. Pohl, and P. Wallen. 1982. Isolation of two variants of native one-chain tissue plasminogen activator. FEBS Lett. 146: 289-292. https://doi.org/10.1016/0014-5793(82)80936-8
  24. Rubin, L. A., C. C. Kurman, M. E. Fritz, W. E. Biddison, B. Boutin, R. Yarchoan, and D. L. Nelson. 1985. Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro. J. Immunol. 135: 3172-3177.
  25. Shohet, R. V., S. Spitzer, E. L. Madison, R. Bassel-Duby, M. J. Gething, and J. F. Sambrook. 1994. Inhibitor-resistant tissuetype plasminogen activator: An improved thrombolytic agent in vitro. Thromb. Haemost. 71: 124-128.
  26. Soleimani, M., F. Mahboudi, N. Davoudi, A. Amanzadeh, M. Azizi, A. Adeli, et al. 2007. Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae. Biotechnol. Appl. Biochem. 48: 55-61. https://doi.org/10.1042/BA20060217
  27. Sporri, B., M. Bickel, D. Dobbelaere, J. Machado Jr., and D. Lottaz. 2001. Soluble interleukin-1 receptor - reverse signaling in innate immunoregulation. Cytokine Growth Factor Rev. 12: 27-32. https://doi.org/10.1016/S1359-6101(00)00020-4
  28. Stettler, M., X. Zhang, D. L. Hacker, J. M. De, and F. M. Wurm. 2007. Novel orbital shake bioreactors for transient production of CHO derived IgGs. Biotechnol. Prog. 23: 1340- 1346. https://doi.org/10.1021/bp070219i
  29. Truelsen, T. and R. Bonita. 2003. Advances in ischemic stroke epidemiology. Adv. Neurol 92: 1-12.
  30. Varley, J. and J. Birch. 1999. Reactor design for large-scale suspension animal cell culture. Cytotechnology 29: 177-205. https://doi.org/10.1023/A:1008008021481
  31. Wagner, O. F., C. de Vries, C. Hohmann, H. Veerman, and H. Pannekoek. 1989. Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA. J. Clin. Invest. 84: 647-655. https://doi.org/10.1172/JCI114211
  32. Weaver, W. D. 1996. The role of thrombolytic drugs in the management of myocardial infarction. Comparative clinical trials. Eur. Heart J. 17 Suppl F: 9-15. https://doi.org/10.1093/eurheartj/17.suppl_F.9
  33. Wurm, F. and A. Bernard. 1999. Large-scale transient expression in mammalian cells for recombinant protein production. Curr. Opin. Biotechnol. 10: 156-159. https://doi.org/10.1016/S0958-1669(99)80027-5
  34. Wurm, F. M. 2004. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22: 1393-1398. https://doi.org/10.1038/nbt1026

피인용 문헌

  1. A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator vol.114, pp.2, 2013, https://doi.org/10.1111/jam.12059
  2. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles vol.19, pp.4, 2015, https://doi.org/10.7508/ibj.2015.04.002
  3. Utilization of Site-Specific Recombination in Biopharmaceutical Production vol.20, pp.2, 2011, https://doi.org/10.7508/ibj.2016.02.001
  4. Proteomics Profiling of Chimeric-Truncated Tissue Plasminogen activator Producing- Chinese Hamster Ovary Cells Cultivated in a Chemically Defined Medium Supplemented with Protein Hydrolysates vol.21, pp.3, 2011, https://doi.org/10.18869/acadpub.ibj.21.3.154
  5. Scale up and pharmacokinetic study of a novel mutated chimeric tissue plasminogen activator (mt-PA) in rats vol.7, pp.None, 2017, https://doi.org/10.1038/srep43028