Skip to main content

Advertisement

SpringerLink
Log in

Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

A poly-Lys tag was fused to the Lucina pectinata hemoglobin I (HbI) coding sequence and purified using an efficient and fast process. HbI is a hemeprotein that binds hydrogen sulfide (H2S) with high affinity and it has been used to understand physiologically relevant reactions of this signaling molecule. The (Lys)6-tagged rHbI construct was expressed in E. coli and purified by immobilization on a cation exchange matrix, followed by size-exclusion chromatography. The identity, structure, and function of the (Lys)6-tagged rHbI were assessed by mass spectrometry, small and wide X-ray scattering, optical spectroscopy, and kinetic analysis. The scattering and spectroscopic results showed that the (Lys)6-tagged rHbI is structurally and functionally analogous to the native protein as well as to the (His)6-tagged rHbI. Kinetics studies with H2S indicated that the association (k on) and dissociation (k off) rate constants were 1.4 × 105/M/s and 0.1 × 10−3/s, respectively. This results confirmed that the (Lys)6-tagged rHbI binds H2S with the same high affinity as its homologue.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Young, C. L., Britton, Z. T., & Robinson, A. S. (2012). Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Biotechnology Journal, 7, 620–634.

    Article  CAS  Google Scholar 

  2. Costa, S., Almeida, A., Castro, A., & Domingues, L. (2014). Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: The novel Fh8 system. Frontiers in Microbiology, 5, 63.

    Google Scholar 

  3. Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M., & Nygren, P. A. (1997). Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expression and Purification, 11, 1–16.

    Article  CAS  Google Scholar 

  4. Terpe, K. (2003). Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 60, 523–533.

    Article  CAS  Google Scholar 

  5. Johnson, C. P., Jensen, I. E., Prakasam, A., Vijayendran, R., & Leckband, D. (2003). Engineered protein a for the orientational control of immobilized proteins. Bioconjugate Chemistry, 14, 974–978.

    Article  CAS  Google Scholar 

  6. Pessela, B. C., Mateo, C., Carrascosa, A. V., Vian, A., Garcia, J. L., Rivas, G., et al. (2003). One-step purification, covalent immobilization, and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads. Biomacromolecules, 4, 107–113.

    Article  CAS  Google Scholar 

  7. Ley, C., Holtmann, D., Mangold, K. M., & Schrader, J. (2011). Immobilization of histidine-tagged proteins on electrodes. Colloids and Surfaces B, 88, 539–551.

    Article  CAS  Google Scholar 

  8. Allard, L., Cheynet, V., Oriol, G., Mandrand, B., Delair, T., & Mallet, F. (2002). Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins. Biotechnology Bioengineering, 80, 341–348.

    Article  CAS  Google Scholar 

  9. Bello-Gil, D., Maestro, B., Fonseca, J., Feliu, J. M., Climent, V., & Sanz, J. M. (2014). Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes. PLoS One, 9, e87995.

    Article  Google Scholar 

  10. Ladaviere, C., Delair, T., Domard, A., Novelli-Rousseau, A., Mandrand, B., & Mallet, F. (1998). Covalent immobilization of proteins onto (Maleic anhydride-alt-methyl vinyl ether) copolymers: enhanced immobilization of recombinant proteins. Bioconjugate Chemistry, 9, 655–661.

    Article  CAS  Google Scholar 

  11. Kweon, D. H., Kim, S.-G., Han, N.-S., Lee, J.-H., Chung, K. M., & Seo, J.-H. (2005). Immobilization of Bacillus macerans cyclodextrin glycosyltransferase fused with poly-lysine using cation exchanger. Enzyme and Microbial Technology, 36, 571–578.

    Article  CAS  Google Scholar 

  12. Kweon, D. H., Lee, D. H., Han, N. S., Rha, C. S., & Seo, J. H. (2002). Characterization of polycationic amino acids fusion systems for ion-exchange purification of cyclodextrin glycosyltransferase from recombinant Escherichia coli. Biotechnology Progress, 18, 303–308.

    Article  CAS  Google Scholar 

  13. Li, J., Zhang, Y., Shen, F., & Yang, Y. (2012). Comparison of magnetic carboxymethyl chitosan nanoparticles and cation exchange resin for the efficient purification of lysine-tagged small ubiquitin-like modifier protease. Journal of Chromatography B, 907, 159–162.

    Article  CAS  Google Scholar 

  14. Nock, S., Spudich, J. A., & Wagner, P. (1997). Reversible, site-specific immobilization of polyarginine-tagged fusion proteins on mica surfaces. FEBS Letters, 414, 233–238.

    Article  CAS  Google Scholar 

  15. Brewer, S. J., & Sassenfeld, J. M. (1985). The purification of recombinant proteins using C-terminal polyarginine fusions. Trends in Biotechnology, 3, 119–122.

    Article  CAS  Google Scholar 

  16. Stempfer, G., Holl-Neugebauer, B., & Rudolph, R. (1996). Improved refolding of an immobilized fusion protein. Nature Biotechnology, 14, 329–334.

    Article  CAS  Google Scholar 

  17. Kraus, D. W., & Wittenberg, J. B. (1990). Hemoglobins of the Lucina pectinata/bacteria symbiosis. I. Reactions with ligands. Journal of Biological Chemistry, 265, 16043–16053.

    CAS  Google Scholar 

  18. Pietri, R., Roman-Morales, E., & Lopez-Garriga, J. (2011). Hydrogen sulfide and hemeproteins: Knowledge and mysteries. Antioxidants & Redox Signaling, 15, 393–404.

    Article  CAS  Google Scholar 

  19. Pietri, R., Lewis, A., Leon, R. G., Casabona, G., Kiger, L., Yeh, S. R., et al. (2009). Factors controlling the reactivity of hydrogen sulfide with hemeproteins. Biochemistry, 48, 4881–4894.

    Article  CAS  Google Scholar 

  20. Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., & Bolognesi, M. (1994). Structure of the sulfide-reactive hemoglobin from the clam Lucina pectinata. Crystallographic analysis at 1.5 A resolution. Journal of Molecular Biology, 244, 86–99.

    Article  CAS  Google Scholar 

  21. Leon, R. G., Munier-Lehmann, H., Barzu, O., Baudin-Creuza, V., Pietri, R., Lopez-Garriga, J., & Cadilla, C. L. (2004). High-level production of recombinant sulfide-reactive hemoglobin I from Lucina pectinata in Escherichia coli. High yields of fully functional holoprotein synthesis in the BLi5 E. coli strain. Protein Expression and Purification, 38, 184–195.

    CAS  Google Scholar 

  22. Steen Redeker, E., Ta, D. T., Cortens, D., Billen, B., Guedens, W., & Adriaensens, P. (2013). Protein engineering for directed immobilization. Bioconjugate Chemistry, 24, 1761–1777.

    Article  CAS  Google Scholar 

  23. Banerjee, R., Chiku, T., Kabil, O., Libiad, M., Motl, N., & Yadav, P. K. (2015). Assay methods for H2S biogenesis and catabolism enzymes. Methods in Enzymology, 554, 189–200.

    Article  Google Scholar 

  24. Luo, H., Zhao, H., Chang, Y., Wang, Q., Yu, H., & Shen, Z. (2015). Oriented immobilization and characterization of a poly-lysine-tagged cephalosporin C acylase on glyoxyl agarose support. Applied Biochemistry and Biotechnology, 175, 2114–2123.

    Article  CAS  Google Scholar 

  25. Chen, Y. H., Chi, M. C., Wang, T. F., Chen, J. C., & Lin, L. L. (2012). Preparation of magnetic nanoparticles and their use for immobilization of C-terminally lysine-tagged Bacillus sp. TS-23 alpha-amylase. Applied Biochemistry and Biotechnology, 166, 1711–1722.

    Article  CAS  Google Scholar 

  26. Collazo, E., Pietri, R., De Jesus, W., Ramos, C., Del Toro, A., Leon, R. G., et al. (2004). Functional characterization of the purified holo form of hemoglobin I from Lucina pectinata overexpressed in Escherichia coli. The Protein Journal, 23, 239–245.

    Article  CAS  Google Scholar 

  27. Antommattei-Perez, F. M., Rosado-Ruiz, T., Cadilla, C. L., & Lopez-Garriga, J. (1999). The cDNA-derived amino acid sequence of hemoglobin I from Lucina pectinata. Journal of Protein Chemistry, 18, 831–836.

    Article  CAS  Google Scholar 

  28. Ramos, C., Pietri, R., Lorenzo, W., Roman, E., Granell, L. B., Cadilla, C. L., & Lopez-Garriga, J. (2010). Recombinant hemoglobin II from Lucina pectinata: A large-scale method for hemeprotein expression in E. coli. The Protein Journal, 29, 143–151.

    Article  CAS  Google Scholar 

  29. Blanchet, C. E., & Svergun, D. I. (2013). Small-angle X-ray scattering on biological macromolecules and nanocomposites in solution. Annual Review of Physical Chemistry, 64, 37–54.

    Article  CAS  Google Scholar 

  30. Mertens, H. D., & Svergun, D. I. (2010). Structural characterization of proteins and complexes using small-angle X-ray solution scattering. Journal of Structural Biology, 172, 128–141.

    Article  CAS  Google Scholar 

  31. Svergun, D. I., Petoukhov, M. V., & Koch, M. H. (2001). Determination of domain structure of proteins from X-ray solution scattering. Biophysical Journal, 80, 2946–2953.

    Article  CAS  Google Scholar 

  32. Svergun, D. I. (1992). Determination of the regularization parameter in indirect-transform methods using perceptual criteria. Journal of Applied Crystallography, 25, 9.

    Article  Google Scholar 

  33. Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40–48.

    Article  Google Scholar 

  34. Roy, A., Kucukural, A., & Zhang, Y. (2010). I-TASSER: A unified platform for automated protein structure and function prediction. Nature Protocols, 5, 725–738.

    Article  CAS  Google Scholar 

  35. Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., & Zhang, Y. (2015). The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12, 7–8.

    Article  CAS  Google Scholar 

  36. McNicholas, S., Potterton, E., Wilson, K. S., & Noble, M. E. (2011). Presenting your structures: the CCP4 mg molecular-graphics software. Acta Crystallographica Section D, 67, 386–394.

    Article  CAS  Google Scholar 

  37. Laspiur, J. P., Anderson, E. R., Ciborowski, P., Wojna, V., Rozek, W., Duan, F., et al. (2007). CSF proteomic fingerprints for HIV-associated cognitive impairment. Journal of Neuroimmunology, 192, 157–170.

    Article  CAS  Google Scholar 

  38. Omenn, G. S., States, D. J., Adamski, M., Blackwell, T. W., Menon, R., Hermjakob, H., et al. (2005). Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database. Proteomics, 5, 3226–3245.

    Article  CAS  Google Scholar 

  39. Fernandez-Alberti, S., Bacelo, D. E., Binning, R. C, Jr, Echave, J., Chergui, M., & Lopez-Garriga, J. (2006). Sulfide-binding hemoglobins: Effects of mutations on active-site flexibility. Biophysical Journal, 91, 1698–1709.

    Article  CAS  Google Scholar 

  40. Nallamsetty, S., & Waugh, D. S. (2007). A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nature Protocols, 2, 383–391.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are most grateful to Prof. Rafael A. Estremera-Andújar for the helpful discussion in the protein purification process, Ms. Ingrid M. Montes-Rodríguez, Mrs. Jessicca Renta, and Dr. José Casanovas-Nieves for their assistance in the molecular cloning analysis as well as undergraduate students Jay J. Rivera Romero and Ruth Martell. The investigators are most grateful to the assistance of Dr. Ariel Lewis-Ballester and Dr. Syun-Ru Yeh for the use of Stopped Flow equipment for kinetic analysis. The support of the RISE 2BEST Program (NIH-RISE NIGMS grant number R25GM088023, RDA), MARC USTAR Program (5T34GM008419-23), the IGERT Nanomedicine Doctoral Fellowship and the Alfred P. Sloan program is deeply appreciated. This project was supported in part by grants from the National Science Foundation (Grant 0843608, JLG), the National Institutes of Health: National Center for Research Resources (G12-RR003051), the National Institute on Minority Health and Health Disparities (G12-MD007600) and Advancing Competitive Biomedical Research in Puerto Rico (P20GM103475).

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Puerto Rico-RUM, Mayagüez, Puerto Rico, USA

    Ramonita Díaz-Ayala, Andrés Moya-Rodríguez, Ruth Pietri & Juan López-Garriga

  2. Department of Biochemistry, University of Puerto Rico Medical Sciences Campus, School of Medicine, San Juan, Puerto Rico, USA

    Carmen L. Cadilla

Authors
  1. Ramonita Díaz-Ayala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrés Moya-Rodríguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ruth Pietri
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carmen L. Cadilla
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan López-Garriga
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juan López-Garriga.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 190 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Díaz-Ayala, R., Moya-Rodríguez, A., Pietri, R. et al. Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata . Mol Biotechnol 57, 1050–1062 (2015). https://doi.org/10.1007/s12033-015-9896-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-015-9896-8

Keywords

Search

Navigation