Purification of human .gamma.-interferon to essential homogeneity and its biochemical characterization

Abstract

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.

Inhibition of endothelial cell proliferation by gamma-interferon.

“…The antiproliferative effects observed with gamma-IFN on endothelial cells in vitro persists for as long as gamma-IFN is present, and upon its removal, the endothelial cells again become responsive to ECGE The effects of gamma-IFN on ECGF-induced HUVEC proliferation could be partially overcome by the addition of 5 U/ml of heparin. Although the mechanism of glycosaminoglycan reversion is not known, gamma-IFN, like ECGF, does bind to heparin (1). Thus, further experiments on the structural interaction between gamma-IFN and heparin are required since it is not clear whether the same glycosaminoglycan in the heparin preparation is responsible for the structural interaction between lymphokine and the growth factor.…”

Glycation and Post-translational Processing of Human Interferon-γ Expressed in Escherichia coli

“…Glycosylation of the protein has been extensively studied (4 -6) and shown to be a source of substantial heterogeneity in natural (7,8) and recombinant hIFN-␥ (rhIFN-␥) preparations (9 -13). Partial proteolysis yielding at least three truncated species further contributes to protein microheterogeneity (14 -16).…”