Oriented Immobilization on Gold Nanoparticles of a Recombinant Therapeutic Zymogen

  • Elina Dosadina
  • Celetia Agyeiwaa
  • William Ferreira
  • Simon Cutting
  • Abdullah Jibawi
  • Enrico Ferrari
  • Mikhail SolovievEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2118)


Direct immobilization of functional proteins on gold nanoparticles (AuNPs) affects their structure and function. Changes may vary widely and range from strong inhibition to the enhancement of protein function. More often though the outcome of direct protein immobilization results in protein misfolding and the loss of protein activity. Additional complications arise when the protein being immobilized is a zymogen which requires and relies on additional protein–protein interactions to exert its function. Here we describe molecular design of a glutathione-S-transferase-Staphylokinase fusion protein (GST-SAK) and its conjugation to AuNPs. The multivalent AuNP-(GST-SAK)n complexes generated show plasminogen activation activity in vitro. The methods described are transferable and could be adapted for conjugation and functional analysis of other plasminogen activators, thrombolytic preparations or other functional enzymes.

Key words

Gold nanoparticles Plasminogen activator Therapeutic protein Protein function Protein stability Experimental design Protein immobilization Staphylokinase Oriented immobilization 


  1. 1.
    Schofield CL, Haines AH, Field RA et al (2006) Silver and gold glyconanoparticles for colorimetric bioassays. Langmuir 22:6707–6711PubMedCrossRefGoogle Scholar
  2. 2.
    Guarise C, Pasquato L, De Filippis V et al (2006) Gold nanoparticles-based protease assay. Proc Natl Acad Sci U S A 103:3978–3982PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Cao YC, Jin R, Mirkin CA (2002) Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297:1536–1540CrossRefGoogle Scholar
  4. 4.
    Park SJ, Taton TA, Mirkin CA (2002) Array-based electrical detection of DNA with nanoparticle probes. Science 295:1503–1506PubMedCrossRefGoogle Scholar
  5. 5.
    Du Y, Luo XL, Xu JJ et al (2007) A simple method to fabricate a chitosan-gold nanoparticles film and its application in glucose biosensor. Bioelectrochemistry 70:342–347PubMedCrossRefGoogle Scholar
  6. 6.
    Boyer D, Tamarat P, Maali A et al (2002) Photothermal imaging of nanometer-sized metal articles among scatterers. Science 297:1160–1163PubMedCrossRefGoogle Scholar
  7. 7.
    Zhang Q, Gong Y, Guo XJ et al (2018) Multifunctional gold nanoparticle-based fluorescence resonance energy-transfer probe for target drug delivery and cell fluorescence imaging. ACS Appl Mater Interfaces 10:34840–34848PubMedCrossRefGoogle Scholar
  8. 8.
    Paciotti GF, Myer L, Weinreich D et al (2004) Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery. Drug Deliv 11:169–183PubMedCrossRefGoogle Scholar
  9. 9.
    Salem AK, Searson PC, Leong KW (2003) Multifunctional nanorods for gene delivery. Nat Mater 10:668–671CrossRefGoogle Scholar
  10. 10.
    Joshi HM, Bhumkar DR, Joshi K et al (2006) Gold nanoparticles as carriers for efficient transmucosal insulin delivery. Langmuir 22:300–305PubMedCrossRefGoogle Scholar
  11. 11.
    Peña B, Maldonado M, Bonham AJ et al (2019) Gold nanoparticle-functionalized reverse thermal gel for tissue engineering applications. ACS Appl Mater Interfaces 11:18671–18680PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    West JL, Halas NJ (2003) Engineered nanomaterials for biophotonics applications: improving sensing, imaging, and therapeutics. Annu Rev Biomed Eng 5:285–292PubMedCrossRefGoogle Scholar
  13. 13.
    Chen R, Riviere JE (2017) Biological and environmental surface interactions of nanomaterials: characterization, modeling, and prediction. WIREs Nanomed Nanobiotechnol 9:e1440CrossRefGoogle Scholar
  14. 14.
    Yin MM, Dong P, Chen WQ et al (2017) Thermodynamics and mechanisms of the interactions between ultrasmall fluorescent gold nanoclusters and human serum albumin, gamma-globulins, and transferrin: a spectroscopic approach. Langmuir 33:5108–5116PubMedCrossRefGoogle Scholar
  15. 15.
    Bailes J, Gazi S, Ivanova R (2012) Effect of gold nanoparticle conjugation on the activity and stability of functional proteins. Methods Mol Biol 906:89–99PubMedGoogle Scholar
  16. 16.
    Lv M, Zhu E, Su Y et al (2009) Trypsin-gold nanoparticle conjugates: binding, enzymatic activity, and stability. Prep Biochem Biotechnol 39(4):429–438PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Huang F, Huang CC, Chang HT (2003) Exploring the activity and specificity of gold nanoparticle-bound trypsin by capillary electrophoresis with laser-induced fluorescence detection. Langmuir 19(18):7498–7502CrossRefGoogle Scholar
  18. 18.
    Zhao X, Hao F, Lu D et al (2015) Influence of the surface functional group density on the carbon-nanotube-induced α-chymotrypsin structure and activity alterations. ACS Appl Mater Interfaces 7:18880–18890PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Pan Y, Neupane S, Farmakes J et al (2017) Probing the structural basis and adsorption mechanism of an enzyme on nano-sized protein carriers. Nanoscale 9:3512–3523PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Halling PJ, Ulijn RV, Flitsch SL (2005) Understanding enzyme action on immobilised substrates. Curr Opin Biotechnol 16:385–392PubMedCrossRefGoogle Scholar
  21. 21.
    Basso A, Braiuca P, Ebert C et al (2006) Properties and applications of supports for enzyme-mediated transformations in solid phase synthesis. J Chem Technol Biotechnol 81:1626–1640CrossRefGoogle Scholar
  22. 22.
    Kolobanova SV, Filippova IY, Lysogorskaya EN (2001) The enzymatic segment condensation of peptides on a solid phase in organic medium. Bioorg Khim 27:347–351PubMedGoogle Scholar
  23. 23.
    Doeze RHP, Maltman BA, Egan CL et al (2004) Profiling primary protease specificity by peptide synthesis on a solid support. Angew Chem Int Ed 43:3138–3141CrossRefGoogle Scholar
  24. 24.
    Cortez J, Vorobieva E, Gralheira D et al (2011) Bionanoconjugates of tyrosinase and peptide-derivatised gold nanoparticles for biosensing of phenolic compounds. J Nanopart Res 13:1101–1113CrossRefGoogle Scholar
  25. 25.
    GE Healthcare Life Sciences (2014) GST Gene Fusion System Handbook. GE Healthcare Life Sciences Protein Purification Methods. Accessed 30 Jan 2018
  26. 26.
    Ferrari E, Darios F, Zhang F et al (2010) Binary polypeptide system for permanent and oriented protein immobilization. J Nanobiotechnology 8:9CrossRefGoogle Scholar
  27. 27.
    Ma W, Saccardo A, Roccatano D et al (2018) Modular assembly of proteins on nanoparticles. Nat Commun 9:1489PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Prasad B, Salunkhe SS, Padmanabhan S (2010) Novel self-cleavage activity of Staphylokinase fusion proteins: an interesting finding and its possible applications. Protein Expr Purif 69:191–197PubMedCrossRefGoogle Scholar
  29. 29.
    Parry MA, Fernandez-Catalan C, Bergner A et al (1998) The ternary microplasmin-Staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action. Nat Struct Biol 5:917–923PubMedCrossRefGoogle Scholar
  30. 30.
    Jespers L, Vanwetswinkel S, Lijnen HR et al (1999) Structural and functional basis of plasminogen activation by Staphylokinase. Thromb Haemost 81:479–485PubMedCrossRefGoogle Scholar
  31. 31.
    Osamu M, Masashi S, Kisaku S et al (1995) Thrombolytic peptide, production thereof, and thrombolytic agent. United States Patent US 5,475,089, 12 Dec 1995Google Scholar
  32. 32.
    Ella KM, Sumathy K (2015) Chimeric Fusion Proteins. United States Patent US 8,968,728, 3 Mar 2015Google Scholar
  33. 33.
    Hoischen C, Gumpert J, Kujau JM et al (2001) Novel l-form bacterial strains, method for producing same and the use thereof for producing gene products. Patent: WO 0166776-A2 13 Sep 2001Google Scholar
  34. 34.
    Wirsching F, Luge C, Schwienhorst A (2002) Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential. Mol Genet Metab 75:250–259PubMedCrossRefGoogle Scholar
  35. 35.
    Szarka SJ, Sihota EG, Habibi HR et al (1999) Staphylokinase as a plasminogen activator component in recombinant fusion proteins. Appl Environ Microbiol 65:506–513PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Yu A, Zhang C, Dong C et al (2008) Two characteristics of a recombinant fusion protein composed of Staphylokinase and hirudin: high thrombus affinity and thrombus-targeting release of anticoagulant activity. Chin J Biotechnol 24:1955–1961CrossRefGoogle Scholar
  37. 37.
    Pulicherla KK, Kumar A, Gadupudi GS et al (2013) In vitro characterization of a multifunctional Staphylokinase variant with reduced reocclusion, produced from salt inducible E. coli GJ1158. Biomed Res Int 2013:297305PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Icke C, Schlott B, Ager OO et al (2002) Fusion proteins with anticoagulant and fibrinolytic properties: functional studies and structural considerations. Mol Pharmacol 62:203–209PubMedCrossRefGoogle Scholar
  39. 39.
    Maheshwari N, Sahni G (2015) Protein fusion constructs possessing thrombolytic and anticoagulant properties. United States Patent US 9,150,844, 6 Oct 2015Google Scholar
  40. 40.
    Hui J, Yu XJ, Cui XJ et al (2014) Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus. Biosci Biotechnol Biochem 78:1514–1521PubMedCrossRefGoogle Scholar
  41. 41.
    Szemraj J, Zakrzeska A, Brown G et al (2011) New derivative of Staphylokinase SAK-RGD-K2-Hirul exerts thrombolytic effects in the arterial thrombosis model in rats. Pharmacol Rep 63:1169–1179PubMedCrossRefGoogle Scholar
  42. 42.
    Wu SC, Castellino FJ, Wong SL (2003) A fast-acting, modular-structured Staphylokinase fusion with Kringle-1 from human plasminogen as the fibrin-targeting domain offers improved clot lysis efficacy. J Biol Chem 278:18199–18206PubMedCrossRefGoogle Scholar
  43. 43.
    Van Zyl WB, Pretorius GH, Lamprecht S et al (2000) PLATSAK, a potent antithrombotic and fibrinolytic protein, inhibits arterial and venous thrombosis in a baboon model. Thromb Res 98:435–443PubMedCrossRefGoogle Scholar
  44. 44.
    Schlott B, Guhrs KH, Hartmann M et al (1997) Staphylokinase requires NH2-terminal proteolysis for plasminogen activation. J Biol Chem 272:6067–6072PubMedCrossRefGoogle Scholar
  45. 45.
    Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40PubMedCrossRefGoogle Scholar
  46. 46.
    Landskroner K, Olson N, Jesmok G (2005) Cross-species pharmacologic evaluation of plasmin as a direct-acting thrombolytic agent: ex vivo evaluation for large animal model development. J Vasc Interv Radiol 16:369–377PubMedCrossRefGoogle Scholar
  47. 47.
    Cederholm-Williams SA (1981) Concentration of plasminogen and antiplasmin in plasma and serum. J Clin Pathol 34:979–981PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Elina Dosadina
    • 1
  • Celetia Agyeiwaa
    • 1
  • William Ferreira
    • 1
  • Simon Cutting
    • 1
  • Abdullah Jibawi
    • 2
  • Enrico Ferrari
    • 3
  • Mikhail Soloviev
    • 4
    Email author
  1. 1.Centre for Biomedical Sciences, School of Biological SciencesRoyal Holloway University of LondonEghamUK
  2. 2.Ashford and St. Peter’s Hospitals NHS Foundation TrustSurreyUK
  3. 3.College of Science, School of Life SciencesUniversity of Lincoln, Brayford PoolLincolnUK
  4. 4.Centre for Biomedical Sciences, Department of Biological SciencesRoyal Holloway University of LondonEghamUK

Personalised recommendations