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Stabilisation of Recombinant Aequorin by Polyols: Activity, Thermostability and Limited Proteolysis

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Abstract

The photoprotein aequorin is a calcium-dependent bioluminescent enzyme which is most widely used in biotechnology processes, but this protein is susceptible to aggregation and proteolysis degradation. Various additives such as polyols are known to enhance the stability of proteins and protect them in native folded and functional state. In this work, for study of aequorin stability, the histidine-tagged apoaequorin was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. Kinetics of light emission of purified aequorin upon addition of Ca2+ showed a linear dependency on aequorin concentration. Furthermore, the effect of some stabilisers, such as glycerol, glucose, lactose, terehalose, sucrose and sorbitol on thermostability of recombinant aequorin was measured. Results indicate that the recombinant aequorin is very stable in phosphate buffer including 30 mM sorbitol, since after heat shock of 30 min at different temperatures, a slight decrease in activity was observed. However, flexibility and exposure of tryptophan residues of aequorin to the solvent, in the presence and absence of stabilisers, with respect to fluorescence quenching by acrylamide, indicated identical characterisation. In addition, according to limited proteolysis of aequorin demonstrating that this enzyme is sensitive to proteases as in the presence of 2 ng/ml of protease, aequorin was completely digested. In conclusion, sorbitol increases stability of aequorin with high photoactivity and not effect for flexibility and limited proteolysis of this photoprotein.

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References

  1. Iyer, P. V., & Ananthanarayan, L. (2008). Process Biochemistry, 43, 1019–1032.

    Article  CAS  Google Scholar 

  2. Stevenson, C. L. (2000). Current Pharmaceutical Biotechnology, 1, 165–182.

    Article  CAS  Google Scholar 

  3. Arakawa, T., & Timasheff, S. N. (1982). Biochemistry, 21, 6536–6544.

    Article  CAS  Google Scholar 

  4. Wetzel, R. (1987). Trends in Biochemical Sciences, 12, 478–482.

    Article  CAS  Google Scholar 

  5. Klibanov, A. M. (1983). Advances in Applied Microbiology, 29, 1–25.

    Article  CAS  Google Scholar 

  6. Jaenicke, R. (2000). Annual Review of Biophysics, 79, 193–203.

    CAS  Google Scholar 

  7. Shimomura, O. (1995). Bid. BdI., 189, 1–5.

    CAS  Google Scholar 

  8. Shimomura, O., Johnson, F. H., & Saiga, Y. (1962). Journal of Cellular and Comparative Physiology, 59, 223–240.

    Article  CAS  Google Scholar 

  9. Head, J. F., Inouye, S., Teranishi, K., & Shimomura, O. (2000). Nature, 405, 372–376.

    Article  CAS  Google Scholar 

  10. Toma, S., Chong, K. T., Nakagawa, A., Teranishi, K., Inouye, S., & Shimomura, O. (2005). Protein Science, 14, 409–416.

    Article  CAS  Google Scholar 

  11. Kawasaki, H., Nakayama, S., & Kretsinger, R. H. (1998). Biometals, 11, 277–295.

    Article  CAS  Google Scholar 

  12. Shimomura, O. (1995). Biochemical and Biophysical Research Communications, 211, 359–363.

    Article  CAS  Google Scholar 

  13. Blinks, J. R. (1990). Environmental Health Perspectives, 84, 75–81.

    Article  CAS  Google Scholar 

  14. Shimomura, O., Musicki, B., & Kishi, Y. (1998). Biochemical Journal, 251, 405–410.

    Google Scholar 

  15. Brini, M., Pinton, P., Pozzan, T., & Rizzuto, R. (1999). Microscopy Research and Technique, 46, 380–389.

    Article  CAS  Google Scholar 

  16. Zerefos, P. G., Ioannou, P. C., & Christopoulos, T. K. (2006). Analytica Chimica Acta, 558, 267–273.

    Article  CAS  Google Scholar 

  17. Christopoulos, T. K., & Galvan, B. (1996). Analytical Chemistry, 68, 3545–3550.

    Article  Google Scholar 

  18. Erikaku, T., Zenno, S., & Inouye, S. (1991). Biochemical and Biophysical Research Communications, 174, 1331–1336.

    Article  CAS  Google Scholar 

  19. Deo, S. K., Lewis, J. C., & Daunert, S. (2000). Analytical Biochemistry, 281, 87–94.

    Article  CAS  Google Scholar 

  20. Baubet, V., Mouellic, H. L., Campbell, A. K., Lucas-Meunier, E., Fossier, P., & Brulet, P. (2000). Proceedings of the National Academy of Sciences, 97(13), 7260–7265.

    Article  CAS  Google Scholar 

  21. Waud, J. P., Bermu, A., Fajardo, D., Sudhaharan, T., Trimby, A. R., Jeffery, J., Jones, A., & Campbell, A. (2001). Biochemical Journal, 357, 687–697.

    Article  CAS  Google Scholar 

  22. Sala-Newby, G. B., Badminton, M. N., Evans, W. H., George, C. H., Jones, H. E., Kendall, J. M., Ribeiro, A. S., & Campbell, A. K. (2000). Methods in Enzymology, 305, 478–498.

    Article  Google Scholar 

  23. Shimomura, O., & Johnson, F. (1969). Biochemistry, 8(10), 3991–3997.

    Article  CAS  Google Scholar 

  24. Inouye, S. (2004). FEBS, 577, 105–110.

    Article  CAS  Google Scholar 

  25. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  26. Laemmli, U. K. (1970). Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  27. Eftink, M. R., & Ghiron, C. A. (1986). Biochemistry, 234, 271–277.

    Google Scholar 

  28. Baimbridge, K. G., Celio, M. R., & Rogers, J. H. (1992). Trends in Neurosciences, 15(8), 303–308.

    Article  CAS  Google Scholar 

  29. Mattson, M. P., & Magnus, T. (2006). Nature Reviews Neuroscience, 7(4), 278–294.

    Article  CAS  Google Scholar 

  30. Inouye, S., Sakaki, Y., Goto, T., & Tsuji, F. I. (1986). Biochemistry, 25, 8425–8429.

    Article  CAS  Google Scholar 

  31. Snowdowne, K. W., & Borle, A. B. (1984). American Journal of Physiology, 247, 396–408.

    Google Scholar 

  32. Fontana, A., Polverino de Laureto, P., Spolaore, B., Frare, E., Picotti, P., & Zambonin, M. (2004). Acta Biochimica Polonica, 51, 299–321.

    CAS  Google Scholar 

  33. Hubbard, S. J. (1998). Biochimica et Biophysica Acta, 1382, 191–206.

    Article  CAS  Google Scholar 

  34. Cioci, F., & Lavecchia, R. (1998). Biotech Technical, 12, 855–858.

    Article  CAS  Google Scholar 

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Acknowledgments

We would like to thank the research council of Mallek-Ashtar and Tarbiat Modares universities for the financial support of this investigation.

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Correspondence to Khosro Khajeh.

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Zeinoddini, M., Khajeh, K., Hosseinkhani, S. et al. Stabilisation of Recombinant Aequorin by Polyols: Activity, Thermostability and Limited Proteolysis. Appl Biochem Biotechnol 170, 273–280 (2013). https://doi.org/10.1007/s12010-013-0096-3

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  • DOI: https://doi.org/10.1007/s12010-013-0096-3

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