SLC15A4 Protein Purification

Pellet from 3.6 L of culture was lyzed with dispersion homogenizer in high salt HEPES-based buffer at pH 7.4, followed by wash and clarification from soluble material at 38.4 kg. Target membrane protein was solubilized for 150 min with 1% of DDM/CHS and clarified by ultracentrifugation at 149 kg. Purification occurs via Strep-affinity batch binding, followed by gravity purification and biotin elution. The SLC15A4 containing fraction were pooled and cleaved with HRV 3C enzyme overnight at +4 °C and finally loaded on SEC column for polishing.

The final and highly pure pool was concentrated at 100 kDa cutoff to ∼1 mg mL^–1, corresponding to yields of ∼0.25 mg/L of culture.

All buffers contained 0.03% DDM (0.006% CHS), and purification steps were carried out at +4 °C.

This material consistently gave, upon NanoDSF Prometheus analysis, a melting temperature of ∼58 °C, with T_m shifts observed upon specific compound addition.