Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.


Basic purification of recombinant HBc protein
To DEPC-treated water. After the addition of RiboLock RNase Inhibitor (Thermo Scientific) to prevent degradation, the mRNA was checked for integrity in formaldehyde agarose gel electrophoresis (FAGE) (see Supplementary Fig. S11c).

For a detailed FAGE protocol, view the TranscriptAid T7 High Yield Transcription
Kit protocol.

Cursory evaluation of VLP nucleic acid (mononucleotide) and protein content
For precise determining of nucleic acid and protein content of viruses and VLPs by UV absorbance can be used general method elaborated by Porterfield and Zlotnick 6 .
In our experiments for routine applications we used simplified calculations based on absorbance ratio at 260 nm and 280 nm for individual componentsnucleotides and empty VLPs, respectively, and also for complexes. Below is an example of the calculations. Ratio A280/A260 for VLPs was as follows: 8 Preparative amounts of PCR fragments were also purified through agarose gel electrophoresis as restriction fragments.
-Single-stranded ONDs were ordered from Metabion and used after reconstitution in water.

Next Generation Sequencing (NGS)
Sequencing was conducted using the Life Technologies Ion Torrent™ PGM platform, which is based on semiconductor sequencing technology 8