Purification Of Human Leukocyte Interferon Research Articles

The human interferon alpha species and receptors.

Interferon (IFN) was approved by the U.S. Food and Drug Administration on June 5, 1986. As the first biotherapeutic approved, IFN-alpha paved the way for development of many other cytokines and growth factors. Nevertheless, we have just touched the surface of understanding the multitude of human IFNs. This paper reviews the history of the purification of human leukocyte IFN and key aspects of our current state of knowledge of human interferon alpha genes, proteins, and receptors.

Editorial: A tribute to Sidney Udenfriend

Editorial: A tribute to Sidney Udenfriend

Purification of human leukocyte interferon A derived from Escherichia coli: An aging process to prevent the formation of its oligomers

Recombinant human leukocyte interferon A (rIFN-αA) was purified from Escherichia coli cells by monoclonal antibody affinity chromatography (Ab) and CM-cellulose column chromatography (CM). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CM eluate was found to contain a large amount of rIFN-αA oligomers, in addition to the monomer, linked by intermolecular disulfide bonds. In contrast, the Ab eluate was found to contain a large amount of rIFN-αA monomers. These results show that the oligomers were formed at the CM step. Free SH groups were detected at a significant level in the Ab eluate, but were detected barely in the CM eluate, indicating that the Ab eluate contained some amount of the unstable monomer with free SH groups. To prevent the formation of the oligomers we established a simple and easily scaled-up process, called an aging process, during which the Ab eluate was incubated at pH 3.3 at 37°C for several days. This process is effective for changing the unstable monomer to the stable monomer and thus preventing the formation of the oligomers.

4485038 Method for the production and purification of human leukocyte interferon: Kailash C Chadha, Eugene Sulkowski assigned to Health Research (Roswell Park Division)

4485038 Method for the production and purification of human leukocyte interferon: Kailash C Chadha, Eugene Sulkowski assigned to Health Research (Roswell Park Division)

Purification of human leukocyte interferon to homogeneity with the use of a monoclonal antibody: Detailed technique and parameters

Summary We had previously described the isolation of anti-human leukocyte interferon monoclonal antibody HBA and its use in the purification and radioimmunoassay of α interferon. The detailed procedure for this purification is described here. Human leukocyte interferon can be purified to homogeneity after two passages on an immunoadsorbant constructed with the monoclonal antibody HBA. The specific activity of the purified interferon is ≥2×10 8 IU/mg of protein. Analysis of the purified proteins by SDS-gel electrophoresis and silver-staining shows that the purified interferon consists of three species of polypeptides of different molecular weights: 18,000 for the major species and 21,000–27,000 for the minor ones. By this simple and rapid technique, it is possible to obtain pure human leukocyte interferon with good recovery (70%) from crude or partially purified preparations. Furthermore, this method could be applied to the large-scale preparation of homogeneous interferon.

Human leukocyte interferon: Isolation and characterization of several molecular forms

Purification of human leukocyte interferon by high-performance liquid chromatography yielded eight major interferon species. These were characterized by their molecular weight, amino acid composition, and antiviral activity on human and bovine cells. The molecular weights of the purified species ranged from 16,000 to 21,000. The tryptic peptide profiles of all the pure species were compared. Both amino acid compositions and tryptic peptide profiles revealed a structural similarity among the various species.

71] Partial purification of human leukocyte interferon on a large scale

Publisher Summary This chapter presents a method for the large-scale purification of human leukocyte interferon. Throughout the purification process, all changes in the pH are made very slowly, while the solutions are stirred briskly. Two batches, concentrated 50-fold and partially purified as described, are routinely pooled for further concentration and purification. The suspensions are thawed in cold water. As soon as the suspension is partly thawed, it is agitated on a magnetic stirrer for 3 hr at 4 °. Insoluble material is removed by centrifugation at 1600 g for 30 min at 4°. The cold interferon is then precipitated at room temperature. The precipitations of the ethanol supernatants are done at 0°C in a refrigerated bath. The pH 8.0 precipitate is dissolved on an orbital shaker for 2 hr and kept overnight at 4°. The precipitations at P-IF A and P-IF B are done at 4 °. The first visible precipitate appears at pH 5.20-5.30. Purified 470 liters of crude interferon is produced after the adoption of these modifications.

69] Purification of human leukocyte interferon by two-dimensional polyacrylamide gel electrophoresis

The homogeneous human leukocyte interferon can be obtained by devising a purification scheme that includes the elimination of heterogeneity by chemical oxidation with sodium periodate, molecular sieve column chromatography, affinity column chromatography, and a modification of the two-dimensional gel electrophoresis technique. A modified two-dimensional gel electrophoresis system is used to further purify the human leukocyte interferon preparations. The modifications made are (1) the omission of reducing agents, such as β-mercaptoethanol, in all solutions and (2), the increased length of the isoelectric focusing gel from l I cm to 24 cm with the corresponding increase of the second-dimension sodium dodecyl sulfate (SDS) gel slab to 35 cm wide by 30 cm in height. Homogeneous spots of two forms of human leukocyte interferons are obtained with this method, each with a final specific activity of about 10 9 units per milligram of protein. This method of two-dimensional gel electrophoresis of periodatetreated preparations can be used to isolate two distinct species of human leukocyte interferon molecules differing in human to bovine activity ratios.

A monclonal antibody for large-scale purification of human leukocyte interferon: Secher, D. S. and Burke, D. C. (1980). Nature, 285, 446

A monclonal antibody for large-scale purification of human leukocyte interferon: Secher, D. S. and Burke, D. C. (1980). Nature, 285, 446

A monoclonal antibody for large-scale purification of human leukocyte interferon

A clone of hybrid myelomas (NK2), secreting a mouse monoclonal antibody to human leukocyte interferon, has been isolated. The antibody neutralizes the antiviral activity of the interferon and, when covalently attached to a solid support and used as an immunoadsorbent, allows interferon purification of up to 5,000-fold in a single step.

Raising antibodies to human leukocyte interferon.

Human leukocyte interferon proved to be a good immunogen in sheep, rabbits and guinea pigs. Sera from immunized animals neutralized the antiviral action of leukocyte interferon at high dilution. The highest anti-interferon titres obtained were 1:1,200,000 for sheep, 1:150,000 for rabbits, 1:30,000 for guinea pigs. Partial purification of human leukocyte interferon, by ethanol precipitation, improved its qualities as an immunogen. While it appeared more efficient to give initial injections without adjuvant, the inclusion in booster injections, of Freund's complete adjuvant produced a markedly superior response and, in one sheep, maintained high levels of circulating antibody for several months.

Production and purification of human leukocyte interferon.

Interferon production in human leukocyte suspensions induced by Newcastle disease virus is significantly affected by the pH of the culture. The optimal pH for interferon production is higher than the physiological pH level. Human leukocyte interferon has at least three types of surface charge as demonstrated by DEAE-cellulose column chromatography. The molecular wight of these three types of interferon, as measured by gel chromatography on several kinds of Sephadex is equally 25 000. The main fraction of human leukocyte interferon was purified approximately 1400-fold by a sequence of procedures which involved ammonium sulfate salting out, CM- and DEAE-cellulose column chromatography and gel filtration on Sephadex G-50. Human leukocyte interferon of a high specific activity of about 2 130 000 units/mg of protein was thus obtained.