Purification of human gamma-interferon to essential homogeneity and its biochemical characterization.
A multistep procedure has been developed which enables human gamma-interferon (HuIFN-gamma) to be purified to essential homogeneity. The procedure takes advantage of a modification of a previously described sequential chromatographic technique [Braude, I.A. (1983) Prep. Biochem. 13, 177-190] and the high isoelectric point of HuIFN-gamma (pH 9.5-9.8). The steps include Controlled Pore Glass adsorption chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, cation-exchange chromatography, and gel filtration chromatography. The purified HuIFN-gamma had a specific activity of 5.9 X 10(7) units/mg. This represents a purification of more than 70 000-fold and a 33% recovery. In addition, one gel filtration fraction had a specific activity of 2.5 X 10(8) units/mg. This represents a purification of greater than 300 000-fold and a recovery of greater than 17%. This fraction, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown to be composed of one major 26-kilodalton (kDa) species and four minor species of 74, 67, 56, and 22 kDa. Analysis of this material with anti-HuIFN-gamma monoclonal antibody immunoabsorbent columns indicates that both the 26- and the 22-kDa species are HuIFN-gamma. Thus, the final product is essentially homogeneous (90-92% HuIFN-gamma), and the specific activity of pure HuIFN-gamma is approximately (2.7-2.8) X 10(8) units/mg of protein. Finally, the 26- and 22-kDa moieties are shown to be similar, if not identical, proteins as judged by amino acid and sequence analyses.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D007163:Immunosorbent Techniques; D007371:Interferon-gamma / Q000097:blood / Q000302:isolation & purification*; D007962:Leukocytes / Q000032:analysis*
D000596:Amino Acids; D007371:Interferon-gamma