Nt 48 nanodsf

DNA constructs encoding SHP1 or SHP2 SH2 domains were cloned into pGMT7 expression vector, transformed into E. coli BL21(DE3)-pLysS (Novagen), expressed as inclusion bodies and refolded using previously described methods (11 (link)). Purification of histidine tagged proteins was performed using FPLC chromatography on HiTrap TALON crude 5 ml column (Cytiva) according to manufacturer’s recommendations. The sample was further purified using size exclusion chromatography on a HiLoad 16/600 Superdex 75 column (Cytiva). The final sample was validated using SDS-PAGE and thermostability assay (Nanotemper NT.48 nanoDSF).

Thermal stability experiments were performed for 0.25–2 mg/ml β4-FNIII-3 and a 20 μM equimolar complex of PRX-C and β4-FNIII-3 in SEC buffer using a label-free fluorescence-based approach (nanoDSF). The instrument used was a NanoTemper Prometheus NT.48 nanoDSF with a backscattering option to detect aggregation onset. Each 10-μl sample was loaded inside a glass capillary, and a constantly monitored scan from 20 to 95°C using a 2°C/min ramp rate was performed. Fluorescence at emission wavelengths 330 nm and 350 nm (F₃₃₀ and F₃₅₀, respectively) was monitored, and a single transition event was observed in the fluorescence ratio (F₃₅₀/F₃₃₀) in all samples. Melting temperature midpoint (T_m) values were extracted from the first derivative peak maximum.