The percentage of protein secondary structures present in the Nc-aD VLPs produced in Sf9 and E. coli was estimated using CD spectroscopy. Purified Nc-aD VLPs (10 µM; 400 µL) were loaded into a quartz cuvette (0.1 cm path length) and scanned with a circular dichroism (CD) spectrometer (JASCO J-815, Japan) at a speed of 100 nm/min at 20 °C. The CD spectrum was collected from wavelengths 190–240 nm. Measurements were done in triplicates. Collected data were analysed using the JASCO secondary structure estimation software [22 (link),45 (link)]. The absorption reading for Nc-aD was corrected by subtracting the absorption reading of the sodium phosphate sample buffer. Measurements of the secondary structures at interval temperatures ranging from 20 to 100 °C were also performed from wavelengths 240 to 190 nm.
To determine the melting temperature (Tm) of Nc-aD VLPs, the samples were prepared as described above for the secondary structure estimation. The samples were subjected to thermal denaturation over a temperature gradient of 20–100 °C, and increment of temperature was maintained at a constant rate of 1 °C/min. Ellipticity and absorbance were recorded using the CD spectrometer (JASCO J-815, Japan), and the spectra were analysed using the JASCO Denaturation Analysis Programme.
To determine the melting temperature (Tm) of Nc-aD VLPs, the samples were prepared as described above for the secondary structure estimation. The samples were subjected to thermal denaturation over a temperature gradient of 20–100 °C, and increment of temperature was maintained at a constant rate of 1 °C/min. Ellipticity and absorbance were recorded using the CD spectrometer (JASCO J-815, Japan), and the spectra were analysed using the JASCO Denaturation Analysis Programme.