Purification and refolding of recombinant human interferon-gamma in urea-ammonium chloride solution.
A method for purification and refolding of recombinant human interferon-gamma (hIFNgamma) 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 hIFNgamma 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 2x10(8)IU/mg; stability - more than two years as a lyophilized powder and more than two months in solution at 4 degrees C.
Petrov
Stefan
S
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria. stefart@abv.bg
Nacheva
Genoveva
G
Ivanov
Ivan
I
D000643:Ammonium Chloride / Q000737:chemistry*; D002460:Cell Line; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D014508:Urea / Q000737:chemistry*
D011994:Recombinant Proteins; D000643:Ammonium Chloride; D007371:Interferon-gamma; D014508:Urea