title: Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins.
abstract: The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom.
authors.forename: Hadrien
authors.initials: H
authors.lastname: Peyret
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: Annick
authors.initials: A
authors.lastname: Gehin
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom.
authors.forename: Eva C
authors.initials: EC
authors.lastname: Thuenemann
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: Donatienne
authors.initials: D
authors.lastname: Blond
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom; iQur Ltd, London, United Kingdom.
authors.forename: Aadil
authors.initials: A
authors.lastname: El Turabi
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom; iQur Ltd, London, United Kingdom.
authors.forename: Lucy
authors.initials: L
authors.lastname: Beales
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: Dean
authors.initials: D
authors.lastname: Clarke
authors.affiliation: UK Division of Structural Biology, University of Oxford, Oxford, United Kingdom.
authors.forename: Robert J C
authors.initials: RJ
authors.lastname: Gilbert
authors.affiliation: UK Division of Structural Biology, University of Oxford, Oxford, United Kingdom.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Fry
authors.affiliation: UK Division of Structural Biology, University of Oxford, Oxford, United Kingdom.
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: Kris
authors.initials: K
authors.lastname: Holmes
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Stonehouse
authors.affiliation: iQur Ltd, London, United Kingdom.
authors.forename: Mike
authors.initials: M
authors.lastname: Whelan
authors.affiliation: iQur Ltd, London, United Kingdom.
authors.forename: William
authors.initials: W
authors.lastname: Rosenberg
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom.
authors.forename: George P
authors.initials: GP
authors.lastname: Lomonossoff
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rowlands
mesh_terms: D019677:Artificial Gene Fusion / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006512:Hepatitis B Core Antigens / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D061905:Single-Domain Antibodies / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D014780:Viruses
chemical_list: D006512:Hepatitis B Core Antigens; D061905:Single-Domain Antibodies; D049452:Green Fluorescent Proteins
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