Recombinant IL-23 Receptor Production

The extracellular part of the IL-23 receptor (IL-23Rex, amino acids 24–350) coded by plasmid DNA (His₆–IL-23Rex–pET-28b [9 (link)] was produced in E. coli SHuffle strain (SHuffle T7 Express Competent E. coli, New England Biolabs, Ipswich, MA, USA). Bacterial cells were grown in LB broth with kanamycin (60 µg/mL) at 30 °C; protein production was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) after the culture reached the density OD₆₀₀ = 0.8, and the cells were cultivated overnight at 23 °C. The next day, the culture was collected by centrifugation and sonicated in TN buffer (50 mM Tris, 300 mM NaCl, pH = 8.0), after which the disrupted cells were spun down at 40,000 g for 20 min and supernatant was used for the purification of soluble protein using Ni-NTA agarose.