Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
권호 표지
DOI QR코드

DOI QR Code

Soluble Expression and Purification of Human Tissue-type Plasminogen Activator Protease Domain

  • Lee, Hak-Joo (Department of Molecular Biology, Sejong University) ;
  • Im, Ha-Na (Department of Molecular Biology, Sejong University)
  • Received : 2010.03.17
  • Accepted : 2010.07.27
  • Published : 2010.09.20
Download PDF
⟨ Previous Next ⟩

Abstract

Human tissue-type plasminogen activator (tPA) is a valuable thrombolytic agent used to successfully treat acute myocardial infarction, thromboembolic stroke, peripheral arterial occlusion, and venous thromboembolism. Recombinant tPA is accumulated as an inactive form in inclusion bodies of E. coli and is refolded in vitro, which is accompanied by extensive aggregation. In the present study, a tPA protease domain was expressed in an active soluble form in the cytosol of E. coli Rosetta-gami cells, which allowed disulfide bond formation and supplied the tRNA molecules required for six rarely used codons in E. coli. This strategy increased the amount of soluble protease domain protein and avoided the cumbersome refolding process. The purified protease domain not only degraded tPA substrate peptides but also formed a covalently bound complex with plasminogen activator inhibitor-1, as does full-length tPA. Soluble expression and purification of tPA domains may aid in functional analyses of this multi-domain protein, which has been implicated in many physiological and pathological processes.

Keywords

References

  1. Rouf, S. A.; Moo-Young, M.; Chisti, Y. Biotechnol. Adv. 1996, 14, 239. https://doi.org/10.1016/0734-9750(96)00019-5
  2. Collen, D.; Lijnen, H. R. Arterioscler. Thromb. Vasc. Biol. 2009, 29, 1151. https://doi.org/10.1161/ATVBAHA.108.179655
  3. Rijken, D. C.; Wijngaards, G.; Zaal-de Jong, M.; Welbergen, J. Biochim. Biophys. Acta 1979, 580, 140. https://doi.org/10.1016/0005-2795(79)90205-8
  4. Collen, D.; Stassen, J. M.; Marafino, B. J.; Builder, S.; DeCock, F.; Ogez, J.; Tajiri, D.; Pennica, D.; Bennett, W. F.; Salwa, J. J. Pharmacol. Exp. Ther. 1984, 231, 146.
  5. Lijnen, H. R.; Collen, D. Thromb. Haemost. 1991, 66, 88.
  6. Allen, S.; Naim, H. Y.; Bulleid, N. J. J. Biol. Chem. 1995, 270, 4797. https://doi.org/10.1074/jbc.270.9.4797
  7. Locker, J. K.; Griffiths, G. J. Cell Biol. 1999, 14, 267.
  8. Jonda, S.; Huber-Wunderlich, N.; Glockshuber, R.; Mossner, E. EMBO J. 1999, 18, 3271. https://doi.org/10.1093/emboj/18.12.3271
  9. Bessette, P. H.; Aslund, F.; Beckwith, J.; Geortiou, G. Proc. Natl. Acad. Sci. USA 1999, 96, 13703. https://doi.org/10.1073/pnas.96.24.13703
  10. Robinson, M.; Lilley, R.; Little, S.; Emtage, J. S.; Yarranton, G.; Stephens, P.; Millican, A.; Eaton, M.; Humphreys, G. Nucleic Acids Res. 1984, 12, 6663. https://doi.org/10.1093/nar/12.17.6663
  11. Ikemura, T. J. Mol. Biol. 1981, 146, 1. https://doi.org/10.1016/0022-2836(81)90363-6
  12. Bringmann, U.; Mattes, R. E.; Buckel, P. Gene 1989, 85, 109. https://doi.org/10.1016/0378-1119(89)90470-8
  13. Pennica, D.; Holmes, W. E.; Kohr, W. J.; Harkins, R. N.; Vehar, G. A.; Ward, C. A.; Bennett, W. F.; Yelverton, E.; Seeburg, P. H.; Heyneker, H. L.; Goeddel, D. V.; Collen, D. Nature 1983, 301, 214. https://doi.org/10.1038/301214a0
  14. Wallen, P.; Pohl, G.; Bergsdorf, N.; Ranby, M.; Ny, T.; Jornvall, H. Eur. J. Biochem. 1983, 1132, 681.
  15. Zhang, Y.; Kanaho, Y.; Frohman, M. A.; Tsirka, S. E. J. Neurosci. 2005, 25, 1797. https://doi.org/10.1523/JNEUROSCI.4850-04.2005
  16. Kim, Y. H.; Park, J. H.; Hong, S. H.; Koh, J. Y. Science 1999, 284, 647. https://doi.org/10.1126/science.284.5414.647
  17. Rogove, A. D.; Siao, C.; Keyt, B.; Strickland, S.; Tsirka, S. E. J. Cell. Sci. 1999, 112, 4007.
  18. Lee, H.-Y.; Hwang, I.-Y.; Im, H.; Koh, J.-Y.; Kim, Y.-H. J. Neurochem. 2007, 101, 1236. https://doi.org/10.1111/j.1471-4159.2007.04417.x
  19. Lim, P. S.; Kwon, M. J.; Kim, S. K.; Nam, S. W. J. Microbiol. Biotech. 2004, 14, 216.
  20. Weickert, M. J.; Pagratis, M.; Curry, S. R.; Blackmore, R. Appl. Environ. Microbiol. 1997, 63, 4313.
  21. Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70. https://doi.org/10.1016/0003-9861(59)90090-6
  22. Baek, J. H.; Im, H.; Kang, U. B.; Seong, K. M.; Lee, C.; Kim, J.; Yu, M. H. Protein Sci. 2007, 16, 1842. https://doi.org/10.1110/ps.072911607
  23. Vaughan, D. E. J. Invest. Med. 1998, 46, 370.
  24. Manosroi, J.; Tayapiwatana, C.; Gotz, F.; Werner, R. G.; Manosroi, A. Appl. Environ. Microbiol. 2001, 67, 2657. https://doi.org/10.1128/AEM.67.6.2657-2664.2001
  25. Mattes, R. Semin. Thromb. Hemost. 2001, 27, 325. https://doi.org/10.1055/s-2001-16886
  26. Vindigni, A.; Cera, E. D. Protein Sci. 1998, 7, 1728. https://doi.org/10.1002/pro.5560070807
  27. Hua, Z.-C. Biochem. Mol. Biol. Intl. 1997, 41, 815.
  28. Wilhelm, O. G.; Jaskunas, S. R.; Vlahos, C. J.; Bang, N. U. J. Biol. Chem. 1990, 265, 14606.

Cited by

  1. vol.43, pp.7, 2013, https://doi.org/10.1080/10826068.2013.764896
  2. Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter vol.10, pp.1, 2015, https://doi.org/10.17795/jjnpp-16676
  3. Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli vol.15, pp.1, 2015, https://doi.org/10.1186/s12896-015-0127-y
  4. Reteplase: Structure, Function, and Production vol.8, pp.None, 2010, https://doi.org/10.4103/abr.abr_169_18
  5. Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator vol.2, pp.2, 2010, https://doi.org/10.3390/mps2020052
  6. Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy vol.8, pp.None, 2010, https://doi.org/10.3389/fmolb.2021.680397