Oligomers of hepatitis A virus (HAV) capsid protein VP1 generated in a heterologous expression system

Fig. 1. Expression and Purification of His-GST-VP1 from E.coli, A His-GST-VP1 purified using nickel chelation chromatography analyzed on 8% SDS-PAGE. Lane 1 represents the protein markers, and lanes 2–4 represent protein fractions eluted with 350 mM imidazole. B Elution profile of His-GST-VP1 from a Superdex 200 (10/300) size exclusion column. C Purified protein from size exclusion chromatography analyzed on 10% SDS-PAGE. Lane 1 represents the protein markers, and lane 2 represents the peak fraction. D Western blots showing the cross reactivity of the purified protein with anti-His (left) and anti-GST (right) antibodies. E and F shows His-GST-VP1 multimers visualized by negative stain and cryo transmission electron microscopy respectively. G shows a 2D class average of His-GST-VP1 multimers from cryo-micrographs

Fig. 2. Expression and purification of His-VP1 under denaturing conditions. A Soluble and pellet fractions of bacterial cells expressing His-VP1 analyzed on 10% SDS-PAGE. Lane 1 represents the protein markers, lane 2 and 3 represents the cellular protein fractions before and after IPTG induction, lane 4 represents the supernatant containing E.coli soluble proteins, and lane 5 represents the insoluble fraction containing His-VP1 B His-VP1 purified using nickel chelation chromatography under denaturing conditions analyzed on 10% SDS-PAGE. Lane 1 represents the protein markers, and lanes 2–8 represent protein fractions eluted with 350 mM imidazole. C Elution profile of His-GST-VP1 from a Superdex 200 (10/300) size exclusion column. D Purified protein from size exclusion chromatography analyzed on 10% SDS-PAGE. Lane 1 represents the protein markers, and lanes 2 and 3 represent the peak fraction. E Western blot showing the cross reactivity of the purified protein against an anti-HAV polyclonal antibody. F LC–MS/MS analysis showing mass spectra of trypsin digested VP1. G Peptide mass fingerprinting and database search showing the presence of HAV VP1

Fig. 3. Size distribution, secondary structure and antigenicity of refolded VP1. A Dynamic Light Scattering (DLS) of refolded VP1, with the Rhmean, Rhpeak and peak area percentage in the inset table. B Circular dichroism (CD) spectra of refolded VP1 with a table showing the percentage of secondary structure components as calculated by the Spectra Manager software of JASCO. C The cross reactivity of refolded VP1 (blue) and recombinantly generated HAV non-structural protein 3Cpro (orange) against a polyclonal anti-HAV antibody analyzed by ELISA. The graph represents the average OD450 for three different concentrations of VP1 and 3Cpro. Values are represented as mean ± SD

Fig. 4. Morphological analysis of refolded VP1 using electron microscopy. A A micrograph of negatively stained refolded VP1, showing the top and side orientations highlighted in yellow and red circles respectively. (Scale bar 100 nm, Magnification 50k×). B 2D classification of refolded VP1 showing 10 different classes with both top (yellow) and side (red) views

Fig. 5. Semi-quantitative analysis of refolded VP1 using SoftEM. A Manually picked particles for 2D classification, B particles picked using SoftEM, C size distribution of particles based on their equivalent radii in nm. D–F show particles with equivalent radii within 5–6 nm, 6–7 nm, and 7–8 nm picked in red, cyan and yellow respectively, which have been quantified in C