Purification and refolding of recombinant human interferon-gamma in urea–ammonium chloride solution

A method for purification and refolding of recombinant human interferon-gamma (hIFNγ) from inclusion bodies is described. It includes the following steps: (i) solubilization of inclusion bodies in 7.4 M guanidinium hydrochloride; (ii) purification of the denatured hIFNγ by hydrophobic chromatography on Octyl-Sepharose column (one step elution with 6 M urea/1 M ammonium chloride); (iii) refolding of the partly purified protein in 0.75 M urea, 20 mM Tris–HCl, pH 8.2; (iv) purification of the refolded protein by CM-Sepharose chromatography. The protein thus obtained is characterized by the following general parameters: yield 1.0 mg/g wet cell mass; purity >99%; specific activity 2 × 108 IU/mg; stability – more than two years as a lyophilized powder and more than two months in solution at 4 °C.