Purification of two subspecies of human gamma (immune) interferon.

Abstract

Interferon (IFN)-y was produced in cultures of human leukocytes by combined stimulation with 12-0-tetradecanoylphorbol 13-acetate (TPA) and phytohemagglutinin (PHA).IFN-ywas purified by sequential adsorption and elution from controlled-pore glass and concanavalin A-Sepharose and by subsequent adsorptive removal of contaminating proteins on DEAESephacel at pH 8.0. Treatment of such partially purified IFN-y preparations with the anionic detergent NaDodSO4 (0.1% at 20-250C) decreased biological activity to approximately 5-20%.When analyzed by NaDodSO4polyacrylamide gel electrophoresis the bulk of IFN activity not destroyed by NaDodSO4 treatment was recovered from two peaks with apparent molecular weights of 20,000 and 25,000. The two activity peaks showed close correspondence with Coomassie blue-stained bands regularly demonstrable in purified supernatants from induced cultures but absent from culture supernatants from uninduced cells. Available evidence suggests that the two bands, isolated in pure form, represent subspecies of IFN-y. Native IFN-y was found to have a lower affinity for alkyl agarose columns than human IFN-a or IFN-fi did, suggesting that IFN-y is a relatively hydrophilic protein. Sulfhydryl-specific binding of native IFN-y to an Affi-Gel 501 column suggested that this IFN contains free sulfhydryl.Until recently virtually no information was available about physicochemical properties ofthe interferon (IFN) species produced in human lymphocytes upon stimulation with specific antigens (1) or with various other mitogens (2, 3). The identification of mitogen-induced IFN as IFN-y and its differentiation from the other two major species, termed IFN-a and IFN-/3 (4), is based mainly on antigenic differences among the three IFN species and on the known relative instability of IFN-y at pH 2 (3).The scarcity of information about IFN-y was mainly due to the lack of suitable methods for its production and purification. Recently, we reported a method of human IFN-y production based on combined stimulation of human lymphocytes with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA) (5, 6). This method enables the production of IFN-y preparations with relatively high initial specific activity. The use of lymphocyte-rich plateletpheresis residues as the cell source for production of IFN-y makes it possible to prepare batches yielding several liters of starting material for purification. We showed earlier that a relatively simple three-step purification procedure led to approximately a 1,000-fold purification of IFN from this starting material (7).Another factor that contributed to the successful purification of IFN-y described in this paper was the recent demonstration that the biological activity of human IFN-y was not completely destroyed by treatment with NaDodSO4 (8). Residual biological activity after NaDodSO4 treatment could thus be used as a marker for the identification of IFN-y proteins under conditions precluding noncovalent interactions am...

Monoclonal antibodies against murine gamma interferon.

“…Indeed, it has been shown that IFN-y molecule produced by mitogen-stimulated T lymphocytes undergoes glycosylation (26), and a number of investigators have shown that mouse IFN-y derived from normal spleen cells stimulated with mitogens displays a broad charge heterogeneity and different degrees of glycosylation and hydrophobicity (26,30,31). Similar findings have also been reported for the human IFN-y by Yip et al (10,32). In particular, they found that IFN--y activity recovered from a partially purified preparation of human IFN-y run on NaDodSO4/PAGE corresponded to two bands with molecular weights of 21 and 25 kDa.…”

“…A limit of this kind of technology, however, is that it does not take into account the possible posttranslational modifications of the synthesized molecules. In fact, as far as IFN-y is concerned, discrepancies on molecular weights have been reported in studies using conventional physicochemical techniques for purification (10) as opposed to those using recombinant DNA technology (7)(8)(9).…”

Constitutive production of human interferons by mouse cells with bovine papillomavirus as a vector.

“…from the deduced amino acid sequence (Mr 17,100) (9) but is similar to the size of natural HuIFN-y produced by peripheral blood lymphocytes (37). This result and the behavior on the Con A-Sepharose column suggest that the IFN-y produced by the transformed line Fy56 contains carbohydrate moieties.…”

Biosynthesis and N‐glycosylation of Human Interferon‐γ

“…In gel analysis, glycosylated IFN-y is typically seen as three bands of different molecular masses independent of its mode of production [9,14,361. These forms represent differential glycosylation of the two N-glycosylation sites.…”