A rapid single-step purification method for human interferon-gamma from isolated Escherichia coli inclusion bodies.
A fast purification method for recombinant human interferon-gamma, produced in E. coli, was elaborated. Human IFN-gamma accumulated in the cytoplasm of E. coli cells as inclusion bodies (IB). After lysis, the IB were isolated from the cell debris by means of a density gradient ultracentrifugation, and solubilized in 6 M guanidine hydrochloride. The subsequent refolding step was optimized for a maximal recovery of the biologically active dimer. Refolded IFN-gamma was then purified to homogeneity in a single cation exchange chromatographic step, yielding 12.5 mg protein per liter E. coli culture. The dimeric nature of the refolded protein was visualized by means of interchain cross-linking. In a subsequent Western blot the resulting derivative was recognized by a panel of five monoclonal antibodies, indicating that those epitopes on the protein surface remained unaffected upon cross-linking.
Haelewyn
J
J
Laboratory of Biochemistry, Katholieke Universiteit Leuven, Belgium.
De Ley
M
M
D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D002479:Inclusion Bodies / Q000378:metabolism*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D017510:Protein Folding; D011994:Recombinant Proteins; D012995:Solubility; D013997:Time Factors
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D007371:Interferon-gamma