Skip to main content
SpringerLink
Log in

New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called “tumor antagonizing genes,” whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression.

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. D’Alessio, G., & Riordan, J. F. (1997). Ribonucleases: Structures and functions. New York: Academic Press.

    Google Scholar 

  2. Li, W. M., Barnes, T., & Lee, C. H. (2010). Endoribonucleases-enzymes gaining spotlight in mRNA metabolism. The FEBS Journal, 277(3), 627–641. doi:10.1111/j.1742-4658.2009.07488.x.

    Article  CAS  Google Scholar 

  3. Luhtala, N., & Parker, R. (2010). T2 Family ribonucleases: ancient enzymes with diverse roles. Trends in Biochemical Sciences, 35(5), 253–259. doi:10.1016/j.tibs.2010.02.002.

    Article  CAS  Google Scholar 

  4. Campomenosi, P., Salis, S., Lindqvist, C., Mariani, D., Nordström, T., Acquati, F., & Taramelli, R. (2006). Characterization of RNASET2, the first human member of the Rh/T2/S family of glycoproteins. Archives of Biochemistry and Biophysics, 449(1–2), 17–26. doi:10.1016/j.abb.2006.02.022.

    Article  CAS  Google Scholar 

  5. Thorn, A., Steinfeld, R., Ziegenbein, M., Grapp, M., Hsiao, H.-H., Urlaub, H., & Krätzner, R. (2012). Structure and activity of the only human RNase T2. Nucleic Acids Research, 40(17), 8733–8742. doi:10.1093/nar/gks614.

    Article  CAS  Google Scholar 

  6. Acquati, F., Possati, L., Ferrante, L., Campomenosi, P., Talevi, S., Bardelli, S., & Taramelli, R. (2005). Tumor and metastasis suppression by the human RNASET2 gene. International Journal of Oncology, 26(5), 1159–1168.

    CAS  Google Scholar 

  7. Acquati, F., Bertilaccio, S., Grimaldi, A., Monti, L., Cinquetti, R., Bonetti, P., & Taramelli, R. (2011). Microenvironmental control of malignancy exerted by RNASET2, a widely conserved extracellular RNase. Proceedings of the National Academy of Sciences of the United States of America, 108(3), 1104–1109. doi:10.1073/pnas.1013746108.

    Article  CAS  Google Scholar 

  8. Acquati, F., Lualdi, M., Bertilaccio, S., Monti, L., Turconi, G., Fabbri, M., & Taramelli, R. (2013). Loss of function of Ribonuclease T2, an ancient and phylogenetically conserved RNase, plays a crucial role in ovarian tumorigenesis. Proceedings of the National Academy of Sciences of the United States of America, 110(20), 8140–8145. doi:10.1073/pnas.1222079110.

    Article  CAS  Google Scholar 

  9. Campomenosi, P., Cinquetti, R., Tallarita, E., Lindqvist, C., Raimondi, I., Grassi, P., & Acquati, F. (2011). Comparison of the baculovirus-insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2. Biotechnology and Applied Biochemistry, 58(1), 39–49. doi:10.1002/bab.7.

    Article  CAS  Google Scholar 

  10. Gietz, R. D., Schiestl, R. H., Willems, A. R., & Woods, R. A. (1995). Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast (Chichester, England), 1(4), 355–360. doi:10.1002/yea.320110408.

    Article  Google Scholar 

  11. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M., Appel, R., & Bairoch, A. (2005). Protein Identification and Analysis Tools on the ExPASy Server. In J. Walker (A C. Di), The Proteomics Protocols Handbook (pp. 571–607). Humana Press. Recuperato da http://dx.doi.org/10.1385/1-59259-890-0%3A571.

  12. Wang, Q., Jiang, M., Wu, J., Ma, Y., Li, T., Chen, Q., & Xiang, L. (2014). Stress-induced RNASET2 overexpression mediates melanocyte apoptosis via the TRAF2 pathway in vitro. Cell Death and Disease, 5, e1022. doi:10.1038/cddis.2013.539.

    Article  CAS  Google Scholar 

  13. Ilinskaya, O. N., & Makarov, A. A. (2005). Why ribonucleases induce tumor cell death. Molecular Biology, 39(1), 1–10. doi:10.1007/s11008-005-0001-4.

    Article  CAS  Google Scholar 

  14. Lee, J. E., & Raines, R. T. (2008). Ribonucleases as novel chemotherapeutics: The ranpirnase example. BioDrugs: Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy, 22(1), 53–58.

    Article  CAS  Google Scholar 

  15. Roiz, L., Smirnoff, P., Bar-Eli, M., Schwartz, B., & Shoseyov, O. (2006). ACTIBIND, an actin-binding fungal T2-RNase with antiangiogenic and anticarcinogenic characteristics. Cancer, 106(10), 2295–2308. doi:10.1002/cncr.21878.

    Article  CAS  Google Scholar 

  16. Schwartz, B., Shoseyov, O., Melnikova, V. O., McCarty, M., Leslie, M., Roiz, L., & Bar-Eli, M. (2007). ACTIBIND, a T2 RNase, competes with angiogenin and inhibits human melanoma growth, angiogenesis, and metastasis. Cancer Research, 67(11), 5258–5266. doi:10.1158/0008-5472.CAN-07-0129.

    Article  CAS  Google Scholar 

  17. Steinfelder, S., Andersen, J. F., Cannons, J. L., Feng, C. G., Joshi, M., Dwyer, D., & Jankovic, D. (2009). The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega-1). The Journal of Experimental Medicine, 206(8), 1681–1690. doi:10.1084/jem.20082462.

    Article  CAS  Google Scholar 

  18. Everts, B., Perona-Wright, G., Smits, H. H., Hokke, C. H., van der Ham, A. J., Fitzsimmons, C. M., & Schramm, G. (2009). Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses. The Journal of Experimental Medicine, 206(8), 1673–1680. doi:10.1084/jem.20082460.

    Article  CAS  Google Scholar 

  19. Everts, B., Hussaarts, L., Driessen, N. N., Meevissen, M. H. J., Schramm, G., van der Ham, A. J., & Yazdanbakhsh, M. (2012). Schistosome-derived omega-1 drives Th2 polarization by suppressing protein synthesis following internalization by the mannose receptor. The Journal of Experimental Medicine, 209(10), 1753–1767. doi:10.1084/jem.20111381.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

F. A. was supported by FAR Insubria University Academic Fund. C. Lindqvist was supported by Åbo Akademi University Foundation. A. I. is grateful for support from the European Research Council and the Associazione Italiana per la Ricerca sul Cancro.

Author information

Author notes

    Authors and Affiliations

    1. Department of Theoretical and Applied Sciences, University of Insubria, Via JH Dunant 3, 21100, Varese, Italy

      Marta Lualdi, Edoardo Pedrini, Debora Scaldaferri, Roberto Taramelli & Francesco Acquati

    2. Laboratory of Immunopharmacology, Humanitas Clinical and Research Center, Via Manzoni 56, 20089, Rozzano, MI, Italy

      Francesca Petroni & Antonio Inforzato

    3. Department of Biosciences, Åbo Akademi University, Tykistokatu 6, 20520, Turku, Finland

      Johnny Näsman & Christer Lindqvist

    Authors
    1. Marta Lualdi
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Edoardo Pedrini
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Francesca Petroni
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Johnny Näsman
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Christer Lindqvist
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Debora Scaldaferri
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Roberto Taramelli
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Antonio Inforzato
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Francesco Acquati
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding authors

    Correspondence to Antonio Inforzato or Francesco Acquati.

    Additional information

    Marta Lualdi and Edoardo Pedrini have contributed equally to the manuscript.

    Electronic Supplementary Material

    Below is the link to the electronic supplementary material.

    12033_2015_9845_MOESM1_ESM.tif

    Supplementary Fig. 1. Comparison of IMAC and IMAC/SEC purification of H65F/H118F_RNASET2-His from BEVS. A) Eluates from IMAC of BEVS cultures expressing H65F/H118F_RNASET2-His (see “Materials and Methods” section) were resolved on a Superdex 75 (10 × 300 mm) column (IMAC, dotted line) into three major fractions (A, B, C). B) These and the unfractionated material (*) were analyzed by SDS-PAGE on 10 % gels under reducing conditions. Representative silver-stained gels are reported (1 μg of total proteins/lane). Fraction C (i.e., that contains H65F/H118F_RNASET2-His) was re-run on SEC (IMAC/SEC, solid line in panel A). (TIFF 681 kb)

    12033_2015_9845_MOESM2_ESM.tif

    Supplementary Fig. 2. Enzymatic deglycosylation of the RNASET2 proteins. Recombinant RNASET2 proteins purified from both P. pastoris and BEVS cultures were deglycosylated with PNGase F and resolved on 13 % Tris–Glycine gels under denaturing and reducing conditions. Proteins were revealed by western blotting with a polyclonal anti-RNASET2 antibody (40 ng of total proteins/lane). Electrophoretic mobility increases as a result of glycan removal from the protein backbone. (TIFF 614 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Lualdi, M., Pedrini, E., Petroni, F. et al. New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris . Mol Biotechnol 57, 513–525 (2015). https://doi.org/10.1007/s12033-015-9845-6

    Download citation

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s12033-015-9845-6

    Keywords

    Search

    Navigation