Multivalent Display Using Hybrid Virus Nanoparticles

  • Steven D. Brown
Part of the Methods in Molecular Biology book series (MIMB, volume 1798)


Many important biological interactions are multivalent and often sensitive to spatial organization. Nonenveloped viruses are a natural source of scaffolds for building multivalent ligands to probe these types of interactions which avoid complex synthetic schemes required for other types of scaffolds. The coat protein (CP) of bacteriophage Qβ can be fused to protein domains and coexpressed with the unfused CP to produce hybrid nanoparticles with high exterior loading of xenogenic protein domains. These hybrid nanoparticles are simple to produce in large quantity. Starting from cDNAs for the virus CP and a codon-optimized ligand domain of interest, bulk purification can be completed in as little as 3 weeks. Major phases of the work involve the cloning of cDNAs into plasmid vectors, test expressions for hybrid nanoparticle formation, and purification by selective precipitation and ultracentrifugation. For uncomplicated protein domains, laboratory culture yields as high as 50 mg/L and 30 protein domains per particle have been routinely achieved.

Key words

Nanoparticle Protein expression Protein purification Multivalency Gene engineering 


  1. 1.
    Mammen M, Choi S-K, Whitesides GM (1998) Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew Chem Int Ed 37(20):2754–2794CrossRefGoogle Scholar
  2. 2.
    Crothers DM, Metzger H (1972) The influence of polyvalency on the binding properties of antibodies. Immunochemistry 9(3):341–357CrossRefPubMedGoogle Scholar
  3. 3.
    Matrosovich MN, Mochalova LV, Marinina VP et al (1990) Synthetic polymeric sialoside inhibitors of influenza virus receptor-binding activity. FEBS J 1(2):209–212CrossRefGoogle Scholar
  4. 4.
    Johansson SM, Arnberg N, Elofsson M et al (2005) Multivalent HSA conjugates of 3′-sialyllactose are potent inhibitors of adenoviral cell attachment and infection. Chembiochem 6(2):358–364. Scholar
  5. 5.
    Zhang L, Qiu W, Crooke S et al (2017) Development of autologous C5 vaccine nanoparticles to reduce intravascular hemolysis in vivo. ACS Chem Biol 12(2):539–547. Scholar
  6. 6.
    Astronomo RD, Kaltgrad E, Udit AK et al (2010) Defining criteria for oligomannose immunogens for HIV using icosahedral virus capsid scaffolds. Chem Biol 17(4):357–370. Scholar
  7. 7.
    Toy R, Roy K (2016) Engineering nanoparticles to overcome barriers to immunotherapy. Bioeng Transl Med 1(1):47–62. Scholar
  8. 8.
    Kaltgrad E, Sen Gupta S, Punna S et al (2007) Anti-carbohydrate antibodies elicited by polyvalent display on a viral scaffold. Chembiochem 8(12):1455–1462. Scholar
  9. 9.
    Lamanna AC, Gestwicki JE, Strong LE et al (2002) Conserved amplification of chemotactic responses through chemoreceptor interactions. J Bacteriol 184(18):4981–4987. Scholar
  10. 10.
    Borrok MJ, Kolonko EM, Kiessling LL (2008) Chemical probes of bacterial signal transduction reveal that repellents stabilize and attractants destabilize the chemoreceptor array. ACS Chem Biol 3(2):101–109CrossRefPubMedGoogle Scholar
  11. 11.
    Handa H, Gurczynski S, Jackson MP et al (2010) Immobilization and molecular interactions between bacteriophage and lipopolysaccharide bilayers. Langmuir 26(14):12095–12103. Scholar
  12. 12.
    Karnik S, Billeter M (1983) The lysis function of RNA bacteriophage Qβ is mediated by the maturation (A2) protein. EMBO J 2(9):1521–1526PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Golmohammadi R, Fridborg K, Bundule M et al (1996) The crystal structure of bacteriophage Qβ at 3.5 angstrom resolution. Structure 4(5):543–554CrossRefPubMedGoogle Scholar
  14. 14.
    Fiedler JD, Higginson C, Hovlid ML et al (2012) Engineered mutations change the structure and stability of a virus-like particle. Biomacromolecules 13(8):2339–2348. Scholar
  15. 15.
    Fuhrmann M, Hausherr A, Ferbitz L et al (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55(6):869–881CrossRefPubMedGoogle Scholar
  16. 16.
    Rosano GL, Ceccarelli EA (2009) Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microb Cell Factories 8:41. Scholar
  17. 17.
    Hoover DM, Lubkowski J (2002) DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 30(10):e43CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hénaut A, Danchin A (1996) Analysis and predictions from Escherichia coli sequences, or E. coli in silico. In: Neidhardt FC (ed) Escherichia coli and Salmonella, vol 2Google Scholar
  19. 19.
    Structural Genomics C, China Structural Genomics C, Northeast Structural Genomics C et al (2008) Protein production and purification. Nat Methods 5(2):135–146. Scholar
  20. 20.
    Oganesyan N, Ankoudinova I, Kim SH et al (2007) Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization. Protein Expr Purif 52(2):280–285. Scholar
  21. 21.
    Blackwell JR, Horgan R (1991) A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS 295(1–3):10–12CrossRefGoogle Scholar
  22. 22.
    Sandee D, Tungpradabkul S, Kurokawa Y et al (2005) Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli. Biotechnol Bioeng 91(4):418–424. Scholar
  23. 23.
    Barth S, Huhn M, Matthey B et al (2000) Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. Appl Environ Microbiol 66(4):1572–1579CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Picaud S, Olsson ME, Brodelius PE (2007) Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli. Protein Expr Purif 51(1):71–79. Scholar
  25. 25.
    Prasad S, Khadatare PB, Roy I (2011) Effect of chemical chaperones in improving the solubility of recombinant proteins in Escherichia coli. Appl Environ Microbiol 77(13):4603–4609. Scholar
  26. 26.
    Schaffner J, Winter J, Rudolph R et al (2001) Cosecretion of chaperones and low-molecular-size medium additives increases the yield of recombinant disulfide-bridged proteins. Appl Environ Microbiol 67(9):3994–4000. Scholar
  27. 27.
    Kusano K, Waterman MR, Sakaguchi M et al (1999) Protein synthesis inhibitors and ethanol selectively enhance heterologous expression of P450s and related proteins in Escherichia coli. Arch Biochem Biophys 367(1):129–136CrossRefPubMedGoogle Scholar
  28. 28.
    Chhetri G, Kalita P, Tripathi T (2015) An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli. MethodsX 2:385–391. Scholar
  29. 29.
    Lobstein J, Emrich CA, Jeans C et al (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Factories 11:56. Scholar
  30. 30.
    Geogiou G, Valax P (1996) Expression of correctly folded proteins in Escherichia coli. Curr Opin Biotechnol 7(2):190–197CrossRefGoogle Scholar
  31. 31.
    Planesse C, Nativel B, Iwema T et al (2015) Recombinant human HSP60 produced in ClearColi BL21(DE3) does not activate the NFkappaB pathway. Cytokine 73(1):190–195. Scholar
  32. 32.
    Brown SD, Fiedler JD, Finn MG (2009) Assembly of hybrid bacteriophage Qβ virus-like particles. Biochemistry 48(47):11155–11157. Scholar
  33. 33.
    Brown SD (2010) Bacteriophage Qβ: a versatile platform for Nanoengineering. The Scripps Research Institute, La Jolla, CAGoogle Scholar
  34. 34.
    Udit AK, Brown S, Baksh MM et al (2008) Immobilization of bacteriophage Qβ on metal-derivatized surfaces via polyvalent display of hexahistidine tags. J Inorg Biochem 102(12):2142–2146. Scholar
  35. 35.
    Udit AK, Hollingsworth W, Choi K (2010) Metal- and metallocycle-binding sites engineered into polyvalent virus-like scaffolds. Bioconjug Chem 21(2):399–404CrossRefPubMedGoogle Scholar
  36. 36.
    TerMaat JR, Pienaar E, Whitney SE et al (2009) Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler. J Microbiol Methods 79(3):295–300. Scholar
  37. 37.
    Xiong AS, Yao QH, Peng RH et al (2006) PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1(2):791–797. Scholar
  38. 38.
    Marsic D, Hughes RC, Byrne-Steele ML et al (2008) PCR-based gene synthesis to produce recombinant proteins for crystallization. BMC Biotechnol 8:44. Scholar
  39. 39.
    Studier FW (2014) Stable expression clones and auto-induction for protein production in E. coli. Methods Mol Biol 1091:17–32. Scholar
  40. 40.
    Liu H, Naismith JH (2008) An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol 8:91. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Steven D. Brown
    • 1
  1. 1.Department of GastroenterologyUniversity of California—San DiegoLa JollaUSA

Personalised recommendations