Skip to main content
SpringerLink
Log in

Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay

  • Paper
  • Published:
Photochemical & Photobiological Sciences Aims and scope Submit manuscript

Abstract

The recombinant coelenterazine-dependent luciferases (isoforms MLuc164 and MLuc39) from the marine copepod Metridia longa were expressed asinclusion bodies in E. coli cells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc39) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free coelenterazine, but also by Ca2+-dependent coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a “substrate” provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35–40 % of the initial activity. The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. G. L. Clarke, R. J. Conover, C. N. David, J. A. Nicol, Comparative studies of luminescence in copepods and other pelagic marine animals Nicol, J. Mar. Biol. Assoc. U.K., 1962, 42, 541–564.

    Article  Google Scholar 

  2. S. V. Markova, S. Golz, L. A. Frank, B. Kalthof, E. S. Vysotski, Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme, J. Biol. Chem., 2004, 279, 3212–3217.

    Article  CAS  Google Scholar 

  3. M. Verhaegen, T. K. Christopoulos, Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization, Anal. Chem., 2002, 74, 4378–4385.

    Article  CAS  Google Scholar 

  4. See, e.g.

  5. B. A. Tannous, D.-E. Kim, J. L. Fernandez, R. Weissleder, X. O. Breakefield, Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo, Mol. Ther., 2005, 11, 435–443.

    Article  CAS  Google Scholar 

  6. K. M. Venisnik, T. Olafsen, S. S. Gambhir, A. M. Wu, Fusion of Gaussia luciferase to an engineered anti-carcinoembryonic antigen (CEA) antibody for in vivo optical imaging, Mol. Imaging Biol., 2007, 9, 267–277.

    Article  Google Scholar 

  7. E. Michelini, L. Ceverini, L. Mezzaonotte, D. Ablamsky, T. Southworth, B. R. Branchini, A. Roda, Combining intracellular and secreted bioluminescent reporter proteins for multicolor cell-based assays, Photochem. Photobiol. Sci., 2008, 7, 212–217.

    Article  CAS  Google Scholar 

  8. M. S. Titushin, S. V. Markova, L. A. Frank, N. P. Malikova, G. A. Stepanyuk, J. Lee, E. S. Vysotski, Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase, Photochem. Photobiol. Sci., 2008, 7, 189–196.

    Article  CAS  Google Scholar 

  9. K. Hori, H. Charbonneau, R. C. Hart, M. J. Cormier, Structure of native Renilla reniformis luciferin, Proc. Natl. Acad. Sci. USA, 1977, 74, 4285–4287.

    Article  CAS  Google Scholar 

  10. B. A. Illarionov, L. A. Frank, V. A. Illarionova, V. S. Bondar, E. S. Vysotski, J. R. Blinks, Recombinant obelin: Cloning and expression of cDNA, purification and characterization as calcium indicator, Methods Enzymol., 2000, 305, 223–249.

    Article  CAS  Google Scholar 

  11. E. S. Vysotski, Z.-J. Liu, J. Rose, B.-C. Wang, J. Lee, Preparation and X-ray crystallographic analysis of recombinant obelin crystals diffracting to beyond 1.1 A, Acta Crystallogr., Sect. D, 2001, 57, 1919–1921.

    Article  CAS  Google Scholar 

  12. L. A. Frank and E. S. Vysotski, Bioluminescent immunoassay for alphafetoprotein using the Ca2+-activated photoprotein obelin, in Bioluminescence & Chemiluminescence: Molecular Reporting with photons, ed. J. W. Hastings, L. J. Kricka and P. E. Stanley, John Wiley, Chichester, 1997, pp. 435–438.

    Google Scholar 

  13. T. E. Creighton, Disulfide bonds between cysteine residues, in Protein structure: a practical approach, ed. T. E. Creighton, IRL Press, Oxford, 1995, pp. 155–167.

    Google Scholar 

  14. R. Jaenicke and R. Rudolph, Folding proteins, in Protein structure: a practical approach, ed. T. E. Creighton, IRL Press, Oxford, 1995, pp. 191–223.

    Google Scholar 

  15. S. M. Singh, A. K. Panda, Solubilization and refolding of bacterial inclusion body proteins, J. Biosci. Bioeng., 2005, 99, 303–310.

    Article  CAS  Google Scholar 

  16. C. Wu, K. Kawasaki, Y. Ogawa, Y. Yoshida, S. Ohgiya, Y. Ohmiya, Preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay, Anal. Chem., 2007, 79, 1634–1638.

    Article  CAS  Google Scholar 

  17. K. Teranishi, O. Shimomura, Coelenterazine analogs as chemiluminescent probe for superoxide anione, Anal. Biochem., 1997, 249, 37–43.

    Article  CAS  Google Scholar 

  18. G. H. E. Law, O. A. Gandelman, L. C. Tisi, C. R. Lowe, J. A. H. Murray, Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance, Biochem. J., 2006, 397, 305–312.

    Article  CAS  Google Scholar 

  19. A. M. Loening, T. D. Fenn, A. M. Wu1, S. S. Gambhir, Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output, Protein Eng. Des. Sel., 2006, 19, 391–400.

    Article  CAS  Google Scholar 

  20. V. Manukhov, G. E. Eroshnikov, M. Yu, Vyssokikh, G. B. Zavilgelsky, Folding and refolding of thermolabile and thermostable bacterial luciferases: the role of DnaKJ heat-shock proteins, FEBS Lett., 1999, 448, 265–268.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Photobiology Lab, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, Russia

    Vasilisa V. Borisova, Ludmila A. Frank, Svetlana V. Markova, Ludmila P. Burakova & Eugene S. Vysotski

  2. Siberian Federal University, pr. Svobodyi, 79, Krasnoyarsk, 660041, Russia

    Ludmila A. Frank

Authors
  1. Vasilisa V. Borisova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ludmila A. Frank
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Svetlana V. Markova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ludmila P. Burakova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eugene S. Vysotski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ludmila A. Frank.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Borisova, V.V., Frank, L.A., Markova, S.V. et al. Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay. Photochem Photobiol Sci 7, 1025–1031 (2008). https://doi.org/10.1039/b807271j

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1039/b807271j

Search

Navigation