Prometheus nanodsf

Protein melting points were determined by differential scanning fluorimetry using both the thermal shift assay and intrinsic tryptophan fluorescence. Thermal shift assay was performed essentially as described earlier. [43 (link)] Briefly, a 50 μl solution containing 1 mg/ml protein in 50 mM Tris, 150 mM NaCl, pH 7.5 (TBS) and 1x SYPRO-Orange (Invitrogen, Carlsbad, CA, USA) was heated in a thermocycler (CFX98, BioRad) from 20°C to 95°C with an approximate heating rate of 1°C/min. Fluorescent readings were taken every 0.5°C using the FRET channel (Ex 450–490, Em 560–580) of the device. T_m was defined as the minima of the first derivative. All data were measured in triplicates. Intrinsic tryptophan fluorescence was measured using a Prometheus NanoDSF (NanoTemper Technologies GmbH, München, Germany) according to the manufacturer protocol. Briefly, protein in MacIlvaine buffer (0.2 M Na₂HPO₄/0.1 M citric acid pH 7.5) was loaded into capillaries and the temperature was increased by 1°C/min from 20°C to 95°C. Intrinsic tryptophan fluorescence was measured at 330 nm and 350 nm and plotted as 350/300 nm ratio. T_m was defined as the minima of the first derivative. All data were measured in duplicates. Analysis of the data was performed using the Origin2018b software.

Thermal shift assays were performed using SYPRO™ orange protein dye (Sigma S5692) at a final dye concentration of 1–5 × and assayed on a Stratagene Mx300 rtPCR for 70 cycles between 25 °C and 95 °C at increments of 2 °C min⁻¹. Fluorescence data was normalised to elucidate the protein melting point (Tm). Thermostability assays were conducted by heat treatment of SMECel6A at 100 °C in various ionic solutions followed by 1h of renaturation at 25 °C. Sequential thermostability and reversible renaturation of SMECel6A was explored in a Prometheus nanoDSF (Nanotemper Technologies, London, UK) at 13.1uM (1mg/mL) in NT.48 standard capillaries during ten thermocycles between 25 °C for 25 min and 90 °C for 5 min at a ramp rate of 1 °C min⁻¹ at pH 5.2 in sodium chloride, artificial seawater and 100 mM citrate phosphate buffer. Denaturation and renaturation were assessed as an index of intrinsic tryptophan fluorescence at 350nm/330nm. Precipitation was assessed as a function of light scattering (mAU). Confirmation of function post heat treatment was validated using the PAHBAH method using 0.5% Avicel™ with 126nM SMECel6A (10 µg mL⁻¹).

Approximately 1 mg mL⁻¹ purified enzyme solution in 100 mM potassium phosphate buffer with 100 mM NaCl (pH 7.2) was loaded into Nano Temper capillary tubes. The capillary tubes were then loaded into a Prometheus nanoDSF (NanoTemper Technologies GmbH, Munich, Germany) and a melting scan with a range from 20 to 95 °C with a heating rate of 1 °C min⁻¹ was performed. The results were evaluated with the NanoTemper analytics software.