Sw32.1 rotor

To prove the expression of recombinant baculovirus, VLPs were produced by infecting Sf-9, Sf-21, or Hi-5 cells cultured in SF-900 II medium with the recombinant baculovirus at a multiplicity of infection (MOI) 5. For large-scale production, Hi-5 cells were cultured in 1-L roller bottles (Corning) with 200 mL working volume. The cells were inoculated at 5 × 10⁷cells, grown to 1 × 10⁸, and then infected with EV71 VLP at MOI 5. At 3 days post-infection (dpi), the infected cells were harvested by centrifugation (10,000 rpm for 10 min), and re-suspended in 0.4μL protease inhibitor (Roche) and benzonase (Novagen) in phosphate buffered saline (PBS) to inhibit proteases during infected cell extractions and preserve the integrity of VLP proteins for further sample characterizations. The cell suspension was then disrupted by sonication and centrifuged at 10,000 rpm for 15 min. Subsequently, the supernatant was ultracentrifuged at 25,000 rpm (SW32.1 rotor, Beckman) for 2 h. The pellets were re-suspended in PBS and loaded onto sucrose gradients (10%, 20%, 30%, 40%, and 50% sucrose dissolved in PBS). After ultracentrifugation at 25,000 rpm for 2 h, the milky white band between the interfaces at 35% sucrose was collected. This fraction was used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE), western blotting, and transmission electron microscopy (TEM) analyses.

Fractionation of polyribosomes was done essentially as described before (9 (link),10 (link)) using 10–50% (17 000 rpm., 18 h) and/or 10–30% (20 000 rpm., 18 h) sucrose gradients and a Beckman SW32.1 rotor. All procedures were performed at 4°C. Yeast cells from 50 ml of log phase culture were pelleted, treated for 10 min with 100 μg/ml cycloheximide and repelleted. Cell extracts were made by glass bead cell disruption (3–5 cycles of 1 min each), with intermittent cooling on ice. The following buffer was used: 100 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES·KOH, pH 7.4, 14.4 mM β-mercaptoethanol, 100 μg/ml cycloheximide. Cell debris was removed by centrifugation at 7000 rpm for 8 min and polyribosomes were resolved by sucrose density gradient centrifugation as indicated. Gradients were collected using the ISCO Programmable Density Gradient System with continuous monitoring at 254 nm using an ISCO UA-6 absorbance detector. Analysis of ratios of 80S monosomes to polyribosomes was done essentially as before (10 (link)). Fractionation of cell extracts using formaldehyde cross-linking was done as described by Nielsen et al. (18 (link)). For western blotting, proteins collected from sucrose gradient fractions were solubilized in sodium dodecyl sulfate (SDS) polyacrylamide sample buffer for 10 min at 95°C, chilled on ice for 5 min and loaded onto polyacrylamide gel.