Analytical and Bioanalytical Chemistry

, Volume 408, Issue 27, pp 7679–7687 | Cite as

Proof of concept of a “greener” protein purification/enrichment method based on carboxylate-terminated carbosilane dendrimer-protein interactions

  • Estefanía González-García
  • Marek Maly
  • Francisco Javier de la Mata
  • Rafael Gómez
  • María Luisa Marina
  • María Concepción García
Research Paper

Abstract

Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample.

Graphical Abstract

Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional “no-clean” method based on acetone precipitation and the proposed “greener” method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio

Keywords

Carboxylate-terminated carbosilane dendrimers Protein sample preparation Protein-dendrimer interaction Fluorescence quenching Computer modeling Molecular dynamics 

Notes

Acknowledgments

This work was supported by the Ministry of Economy and Competitiveness (ref. AGL2012-36362 and CTQ-2014-54004-P), the Comunidad de Madrid and European funding from FEDER program (ref. S2013/ABI-3028, AVANSECAL), and Consortium NANODENDMED ref. S2011/BMD-2351 (CAM). The financial support of the Czech Science Foundation (project no. GA15-05903S) is also acknowledged. E.G.-G. thanks the University of Alcalá for her pre-doctoral contract. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest to declare.

Supplementary material

216_2016_9864_MOESM1_ESM.pdf (1.5 mb)
ESM 1 (PDF 1498 kb)

References

  1. 1.
    Wang W, Tai F, Chen S. Optimizing protein extraction from plant tissues for enhanced proteomics analysis. J Sep Sci. 2008;31(11):2032–9.CrossRefGoogle Scholar
  2. 2.
    Wu X, Gong F, Wang W. Protein extraction from plant tissues for 2DE and its application in proteomic analysis. Proteomics. 2014;14(6):645–58.CrossRefGoogle Scholar
  3. 3.
    Wang W, Vignani R, Scali M, Cresti M. A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis. 2006;27(13):2782–6.CrossRefGoogle Scholar
  4. 4.
    Wongpia A, Mahatheeranont S, Lomthaisong K, Niamsup H. Evaluation of sample preparation methods from rice seeds and seedlings suitable for two-dimensional gel electrophoresis. Appl Biochem Biotechnol. 2015;175(2):1035–51.CrossRefGoogle Scholar
  5. 5.
    Banasiak A. Evolution of the cell wall components during terrestrialization. Acta Soc Bot Pol. 2014;83(4):349–62.CrossRefGoogle Scholar
  6. 6.
    Ward WW, Swiatek G. Protein purification. Curr Anal Chem. 2009;5(2):85–105.CrossRefGoogle Scholar
  7. 7.
    Wisniewski JR, Zielinska DF, Mann M. Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method. Anal Biochem. 2011;410(2):307–9.CrossRefGoogle Scholar
  8. 8.
    Feist P, Hummon AB. Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int J Mol Sci. 2015;16(2):3537–63.CrossRefGoogle Scholar
  9. 9.
    Jiang L, He L, Fountoulakis M. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J Chromatogr A. 2004;1023(2):317–20.CrossRefGoogle Scholar
  10. 10.
    Englard S, Seifter S. Precipitation techniques. In: Deutscher MP, editor. Methods in enzymology, vol. 182. New York: Academic Press, Inc; 1990.Google Scholar
  11. 11.
    Kalhapure RS, Kathiravan MK, Akamanchi KG, Govender T. Dendrimers—from organic synthesis to pharmaceutical applications: an update. Pharm Dev Technol. 2015;20(1):22–40.CrossRefGoogle Scholar
  12. 12.
    Martinho N, Florindo H, Silva L, Brocchini S, Zloh M, Barata T. Molecular modeling to study dendrimers for biomedical applications. Molecules. 2014;19(12):20424–67.CrossRefGoogle Scholar
  13. 13.
    Jiménez JL, Gómez R, Briz V, Madrid R, Bryszewsk M, de la Mata FJ, et al. Carbosilane dendrimers as carriers of siRNA. J Drug Delivery Sci Technol. 2012;22(1):75–82.CrossRefGoogle Scholar
  14. 14.
    Schlenk C, Frey H. Carbosilane dendrimers—synthesis, functionalization, application. Mon Chem. 1999;130(1):3–14.Google Scholar
  15. 15.
    Galán M, Sánchez Rodríguez J, Jimenez JL, Relloso M, Maly M, de la Mata FJ, et al. Synthesis of new anionic carbosilane dendrimers via thiol-ene chemistry and their antiviral behaviour. Org Biomol Chem. 2014;12(20):3222–37.CrossRefGoogle Scholar
  16. 16.
    Wang JM, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of a general amber force field. J Comput Chem. 2004;25(9):1157–74.CrossRefGoogle Scholar
  17. 17.
    Bayly CI, Cieplak P, Cornell WD, Kollman PA. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges—the RESP model. J Phys Chem. 1993;97(40):10269–80.CrossRefGoogle Scholar
  18. 18.
    Dupradeau F, Pigache A, Zaffran T, Savineau C, Lelong R, Grivel N, et al. The R.ED. tools: advances in RESP and ESP charge derivation and force field library building. Phys Chem Chem Phys. 2010;12(28):7821–39.CrossRefGoogle Scholar
  19. 19.
    Gordon MS, Schmidt MW. Advances in electronic structure theory: GAMESS a decade later. In: Frenking G, Dykstra CE, editors. Theory and applications of computational chemistry: the first forty years. Amsterdam: Elsevier; 2005. p. 1167–89.CrossRefGoogle Scholar
  20. 20.
    Bujacz A. Structures of bovine, equine and leporine serum albumin. Acta Crystallogr Sect D: Biol Crystallogr. 2012;68:1278–89.CrossRefGoogle Scholar
  21. 21.
    Botha S, Nass K, Barends TRM, Kabsch W, Latz B, Dworkowski F, et al. Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams. Acta Crystallogr Sect D: Biol Crystallogr. 2015;71:387–97.CrossRefGoogle Scholar
  22. 22.
    Hubbard SR, Hendrickson WA, Lambright DG, Boxer SG. X-ray crystal-structure of a recombinant human myoglobin mutant at 2.8 a resolution. J Mol Biol. 1990;213(2):215–8.CrossRefGoogle Scholar
  23. 23.
    Case DA, Babin V, Berryman JT, Betz RM,. Cai Q, Cerutti DS, et al. AMBER 14. 2014.Google Scholar
  24. 24.
    Goetz AW, Williamson MJ, Xu D, Poole D, Le Grand S, Walker RC. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized born. J Chem Theory Comput. 2012;8(5):1542–55.CrossRefGoogle Scholar
  25. 25.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12.CrossRefGoogle Scholar
  26. 26.
    González-García E, Marina ML, García MC. Plum (Prunus domestica L.) by-product as a new and cheap source of bioactive peptides: extraction method and peptides characterization. J Funct Foods. 2014;11:428–37.CrossRefGoogle Scholar
  27. 27.
    Bradford MM. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem. 1976;72(1–2):248–54.CrossRefGoogle Scholar
  28. 28.
    Eftink MR. The use of fluorescence methods to monitor unfolding transitions in proteins. Biophys J. 1994;66(2):482–501.CrossRefGoogle Scholar
  29. 29.
    Marden MC, Hoa GHB, Stetzkowskimarden F. Heme protein fluorescence versus pressure. Biophys J. 1986;49(3):619–27.CrossRefGoogle Scholar
  30. 30.
    Hashimoto S, Fukasaka J, Takeuchi H. Structural study on acid-induced unfolding intermediates of myoglobin by using UV resonance Raman scattering from tryptophan residues. J Raman Spectrosc. 2001;32(6–7):557–63.CrossRefGoogle Scholar
  31. 31.
    Baler K, Martin OA, Carignano MA, Ameer GA, Vila JA, Szleifer I. Electrostatic unfolding and interactions of albumin driven by pH changes: a molecular dynamics study. J Phys Chem B. 2014;118(4):921–30.CrossRefGoogle Scholar
  32. 32.
    Lee JW, Kim HI. Investigating acid-induced structural transitions of lysozyme in an electrospray ionization source. Analyst. 2015;140(2):661–9.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Estefanía González-García
    • 1
  • Marek Maly
    • 2
  • Francisco Javier de la Mata
    • 3
  • Rafael Gómez
    • 3
  • María Luisa Marina
    • 1
  • María Concepción García
    • 1
  1. 1.Departamento de Química Analítica, Química Física e Ingeniería QuímicaUniversidad de AlcaláAlcalá de HenaresSpain
  2. 2.Faculty of ScienceJ. E. Purkinje UniversityUsti nad LabemCzech Republic
  3. 3.Departamento de Química Orgánica y Química InorgánicaUniversidad de Alcalá, Ctra. Madrid-BarcelonaAlcalá de HenaresSpain

Personalised recommendations