Purification of Human Fibroblast Interferon by Zinc Chelate Chromatography

Abstract

Human interferon [an antiviral glycoprotein] was prepared by superinduction of cultures of either diploid embryonic skin and muscle cells or of the osteosarcoma cell line MG-63. The interferon so obtained was concentrated and partially purified by adsorption to controlled pore glass (CPG) beads at neutral pH, and desorption by glycine-HC1 buffer at pH 2. After neutralization, this interferon was applied to a column of zinc chelate which was eluted with buffers of decreasing pH. Most of the proteins eluted ahead of the interferon activity, which itself eluted in 2 distinct peaks. The 1st peak occurred in the effluent fractions around pH 5.9 and the 2nd one in fractions around pH 5.2. The interferon found in fractions of pH 5.9 contained 5% of the original contaminating proteins. The amount of total protein in the pH 5.2 peak was so small that it could not accurately be assayed by the fluorescamine method. The interferon in the peak fraction was estimated to have a specific activity of about 2 .times. 109 U/mg. This material was radiolabeled and analyzed by electrophoresis. A major peak of about 22,000 MW with only minor contaminating proteins appeared on the autoradiographs. The total recovery of the zinc chelate chromatographical procedure was nearly 100%, and the interferon recovered from each peak behaved consistently on rechromatography. Fibroblast interferon produced by most diploid cells contained < 10% of the variant eluting at pH 5.9. MG-63 cells and high-passage cultures of some diploid cell strains produced up to 50% of this variant.