Skip to main content

Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs)

Part of the Methods in Molecular Biology book series (MIMB,volume 1776)


The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98–114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent “spike.” The tips of the “spikes” are the preferred sites for the insertion of foreign sequences for vaccine purposes as they are the most highly exposed regions of the assembled particles. In the tandem-core modification two copies of the HBc protein are covalently linked by a flexible amino acid sequence which allows the fused dimer to fold correctly and assemble into HBc particles. The advantage of the modified structure is that the assembly of the dimeric subunits is defined and not formed by random association. This facilitates the introduction of single, larger sequences at the tip of each surface “spike,” thus overcoming the conformational clashes contingent on insertion of large structures into monomeric HBc proteins.

Differences in inserted sequences influence the assembly characteristics of the modified proteins, and it is important to optimize the design of each novel construct to maximize efficiency of assembly into regular VLPs. In addition to optimization of the construct, the expression system used can also influence the ability of recombinant structures to assemble into regular isometric particles. Here, we describe the production of recombinant tandem-core particles in bacterial, yeast and plant expression systems.

Key words

  • Hepatitis B core (HBc)
  • HBc VLPs
  • Escherichia coli expression
  • Pichia pastoris expression
  • Plant expression

This is a preview of subscription content, access via your institution.

Buying options

USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-7808-3_7
  • Chapter length: 27 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
USD   189.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-7808-3
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   249.99
Price excludes VAT (USA)
Hardcover Book
USD   249.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. Pumpens P, Grens E (2001) HBV core particles as a carrier for B cell/T cell epitopes. Intervirology 44:98–114

    CrossRef  CAS  PubMed  Google Scholar 

  2. Peyret H, Gehin A, Thuenemann EC, Blond D, El Turabi A, Beales L, Clarke D, Gilbert RJ, Fry EE, Stuart DI, Holmes K, Stonehouse NJ, Whelan M, Rosenberg W, Lomonossoff GP, Rowlands DJ (2015) Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins. PLoS One 10:e0120751

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sainsbury F, Thuenemann EC, Lomonossoff GP (2009) pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7:682–693

    CrossRef  CAS  PubMed  Google Scholar 

  4. Peyret H, Lomonossoff GP (2013) The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants. Plant Mol Biol 83(1–2):51–58

    CrossRef  CAS  PubMed  Google Scholar 

  5. Peyret H (2015) A protocol for the gentle purification of virus-like particles produced in plants. J Virol Methods 225:59–63

    CrossRef  CAS  PubMed  Google Scholar 

  6. Stonehouse NJ, Stockley PG (1993) Effects of amino acid substitution on the thermal stability of MS2 capsids lacking genomic RNA. FEBS Lett 334:355–359

    CrossRef  CAS  PubMed  Google Scholar 

  7. Liljeroos L, Huiskonen JT, Ora A, Susi P, Butcher SJ (2011) Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions. Proc Natl Acad Sci U S A 108:18085–18090

    CrossRef  PubMed  PubMed Central  Google Scholar 

  8. Lua LH, Connors NK, Sainsbury F, Chuan YP, Wibowo N, Middelberg AP (2014) Bioengineering virus-like particles as vaccines. Biotechnol Bioeng 111:425–440

    CrossRef  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to David J. Rowlands .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

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

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Stephen, S.L., Beales, L., Peyret, H., Roe, A., Stonehouse, N.J., Rowlands, D.J. (2018). Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs). In: Wege, C., Lomonossoff, G. (eds) Virus-Derived Nanoparticles for Advanced Technologies. Methods in Molecular Biology, vol 1776. Humana Press, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7806-9

  • Online ISBN: 978-1-4939-7808-3

  • eBook Packages: Springer Protocols