Purification and conformational properties of a human interferon α2b produced in Escherichia coli

Recombinant human interferon a2b was expressed intracellularly in Escherichia coli as insoluble aggregates using a new expression vector, and was purified to homogeneity using essentially two-step chromatographic procedures, i.e. immobilized metal-ion-affinity chromatography and reversed-phase HPLC. The established purification process is highly reproducible and leads to a total recovery of approx. 12% with a specific biological activity of higher than 1 × 10 8 i.u./mg of protein, which is comparable with the international requirement for interferon α2b. For purified protein we report conformational stability as a function of pH and temperature using differential scanning calorimetry and CD. Thermal unfolding as a function of pH showed only one endotherm at a temperature higher than 45 °C, and was reversible at pH 2-3.75 and irreversible at pH 4-10. At pH 7.0, the most stable condition, the conformational stability depends on protein concentration and ionic strength. The highly helical secondary structure is very conserved over the whole pH range studied, including at high temperatures.