Interferon: purification and initial characterization from human diploid cells.

Abstract

Interferon produced by human diploid fibroblast cells in culture has been purified approximately 5000-fold. The purified interferon, when analyzed by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate, contains only one polypeptide component of 20,000 molecular weight. The interferon activity comigrates with this polypeptide, indicating identity of the activity and the polypeptide. Oxidation of this polypeptide with periodic acid and subsequent staining with Fuchsin base indicates that it contains carbohydrate and suggests that the human fibroblast interferon is a glycoprotein.Interferon, a broad-spectrum antiviral protein, has been the subject of numerous biological studies, yet its chemical characterization from any source has remained elusive (see refs. 1 and 2 for reviews). Human leukocyte (3) and human diploid fibroblast (3-5) interferons have been purified by various techniques. This paper reports (i) a procedure for purifying human diploid fibroblast interferon and (ii) some chemical and physical properties of the purified interferon. MATERIALS AND METHODSProduction of Crude Interferon. The human diploid fibroblast cells (neonatal foreskin fibroblasts, FS-4) were a gift from Drs. E. Havell and J. Vilcek. The procedures for growing the cells and producing the interferon have been described (6). For production of interferon, cells were grown at 370 in glass roller bottles 20 cm long and 9.5 cm internal diameter. Induction of interferon with poly(I)-poly(C) was performed exactly as previously described (6). Briefly, cell cultures 10-12 days old, 4 X 107 cells per bottle, were induced, then allowed to produce interferon at 340 for 20 hr in serum-free medium, 50 ml per bottle. Crude interferon was collected and stored at 40 until a sufficient quantity was accumulated for purification. Titers of crude interferon varied from 4000 to 20,000 units/ml. The crude interferon was concentrated 10-to 20-fold prior to purification, as described for mouse L cell interferon (7). Interferon was assayed on FS-4 cells with vesicular stomatitis virus as the challenge virus (6). All units are expressed as reference units.Polyacrylamide Gel Electrophoresis. Preparative electrophoresis was performed in cylindrical separating gels, 1 cm X 20 cm, using the discontinuous buffer system of Laemmli (8) with sodium dodecyl sulfate (NaDodSO4). Stacking gels 2 cm in length of 5% acrylamide were cast on top of the separating gel. The separating gel was an exponential gradient gel (7, 9) of 9-18% acrylamide. The gradient was prepared by mixing 20 ml of 9% acrylamide with 3 ml of 18% acrylamide kept at constant volume, and the ratio of acrylamide to bis-acrylamide was 38:1. All gels and buffers contained 0.1% NaDodSO4. Analytical slab gels, 9-18% acrylamide, were prepared as described (7, 9). Protein in the gels was fixed and stained for 15 min in a solution of methanol:acetic acid:water 50:10:40 (vol/vol/vol) containing 1.25 mg/ml of Coomassie blue R 250. Gels were destained in 7% acetic-5% methanol and dried un...

Purification and Initial Characterization of Human Placental Trophoblast Interferon Induced by Polyriboinosinic {middle dot} Polyribocytidylic Acid

“…6). The Mr of the tro-IFN, however, differed from the 20K value reported by others (Knight & Fahey, 1980;Knight, 1976;Tan et al, 1979) for IFN-fl induced in other human diploid cells but it was the same of the value obtained by Otto et al (1980) for IFN-fl induced in the FS-4 strain of human foreskin fibroblast cells by poly(rI).poly(rC). Other workers (Reynolds & Pitha, 1975;Heine et al 1981;Duc-Goiran et al, 1983) which may have resulted in the differences in their secondary structures.…”

“…We have explored the use of Blue Sepharose affinity chromatography and RP-HPLC on Separon SGX C-18 to isolate a homogeneous tro-IFN of high quality suitable for structural analysis, monoclonal antibody production and biochemical and clinical studies. The purification method described here has several advantages over a number already reported (Knight, 1976;Rubinstein et al, 1978;Okamura et al, 1980) for purifying human IFNs. The method is simple in that it involves only two steps, it yields a homogeneous product, it does not involve extremes of pH and therefore can be used successfully to purify a number of different IFNs (including acid-labile IFNs) with retention of biological activity, and can easily be scaled-up to meet the growing demand for large amounts of pure IFNs.…”

Production and Purification of Human MG63 Cell Interferon

“…poly(C) to a specific activity of2 X 10 9 IU/mg of protein with an overall recovery of about 50%. Other workers (3,19,20,31) obtained values of2-l0 X 10 8 IU/mg of protein as the specific activity of pure human IFN-,8 from poly(I).poly(C)-induced diploid fibroblasts. The differences in the specific activity of pure IFN-,8 found by different workers may be due to the different methods of protein estimation and to varying degrees of inactivation during purification; further work is needed to determine precisely the "true" value of the specific activity.…”

Early interferon production in human diploid cells induced by newcastle disease virus in the presence of protein synthesis inhibitors.

“…It has been reported that human interferon j3 is a glycoprotein (Knight Jr. 1976), and its production has been inhibited effectively by glycosylation inhibitors ). On the other hand, human interferon a is not glycosylated (Mogensen et al 1974 ;Nagata et al 1980), and production of human interferon a has been inhibited by higher concentration of the glycosylation inhibitors than a concentration of inhibitor for human interferon /3 (Stewart II & Stewart 1979).…”