Thermal Stability by Circular Dichroism (CD)

The effects of C4I, C5D, and C5K tags on structure conformation were monitored by CD by dissolving the lyophilized protein powders in PBS, pH 7.4. Before CD measurements, all samples were centrifuged at 20,000 × g for 20 min at 4°C to remove aggregates that might have accumulated during sample preparation. The CD spectra were measured using a 2-mm cuvette with a JASCO J-820 spectropolarimeter at 0.10–0.45 mg/ml concentration in the temperature range 20–90°C and in the wavelength range 200–260 nm. The reversibility of thermal unfolding–refolding was confirmed by measuring the CD of the sample after cooling it back to 20°C from 90°C.