Among the various types of human interferon, immune interferon (IFN-.gamma.) is the least characterized. This interferon is produced in lymphocytes upon stimulation with mitogens or specific antigens and it differs significantly in its structure and properties from the virus-induced .alpha.- and .beta.-interferons.
Recently, Yip et al., have reported a method of human IFN-.gamma. production based on the stimulation of lymphocytes by a combination of the phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate (TPA) and phytohemagglutinin (PHA)(1). Later, Yip et al. were able to purify two subtypes of IFN-.gamma. by a preparative NaDodSO.sub.4 /polyacrylamide gel electrophoresis. However, an almost complete loss of biological activity had occurred during this step (2,3). These subtypes had an apparent molecular mass of 20,000 and 25,000, they corresponded to Coomassie blue-stainable protein bands, and were antigenically cross-reactive. A third minor component having an apparent molecular mass of about 43,000 daltons was also detected.
In another study, Gray et al. have reported the cloning and expression of human IFN-.gamma. complementary DNA. The nucleotide sequence indicated that this IFN-.gamma. was composed of a single polypeptide with 146 amino acids and a calculated molecular mass of 17,000 daltons. Furthermore, analysis of a gene library did not reveal any other structurally related DNA sequences (4). This data suggested that there is only one polypeptide sequence related to IFN-.gamma. and that native IFN-.gamma. may be partly dimeric.
Pestka and Rubinstein have previously described in U.S. Pat. No. 4,289,690 the purification to homogeneity of IFN-.alpha. and the separation of eight distinct subtypes by reverse phase high performance liquid chromatography (HPLC)(5,6,7). This method is not suitable for the purification of IFN-.gamma. because IFN-.gamma. is labile to organic solvents and low pH. The recent availability of ion exchange HPLC columns suitable for fractionation of proteins has allowed high resolution chromatography of IFN-.gamma. preparations.
Recent scientific papers directed to the production, purification and structural studies of IFN-.gamma. can be summarized as follows:
(1) Yip, Y. K., Pang. R. H. L., Urban, C. and Vilcek, J. (1981). Proc.Natl.Acad.Sci. USA 78, 1601-1605. PA1 (2) Yip, Y. K., Barrowclough, B. S., Urban, C. and Vilcek, J. (1982). Science 215, 411-413. PA1 (3) Yip, Y. K., Barrowclough, B. S., Urban, C. and Vilcek, J. (1982). Proc.Natl.Acad.Sci. USA 79, 1820-1824. PA1 (4) Gray, P. W., Leung, D. W., Pennica, D., Yelverton, E., Najarian, R., Simonsen, C. C., Derynck, R., Sherwood, P. J., Wallace, D. M., Berger, S. L., Levinson, A. D. and Goeddel, D. V. (1982). Nature 295, 503-508. PA1 (5) Rubinstein, M., Rubinstein, S., Familletti, P. C., Miller, R. S., Waldman, A. A. and Pestka, S. (1979). Proc.Natl.Acad.Sci. USA 76,640-644. PA1 (6) Rubinstein, M. (1979). Anal. Biochem. 97, 1-7. PA1 (7) Rubinstein, M., Levy, W. P., Moschera, J. A., Lai-C. Y., Hershberg, R. D., Bartlett, R. and Pestka, S. (1981) Arch.Biochem.Biophys. 210, 307-318.
While several of the above papers contain claims to have purified human immune interferons to homogeneity, an almost complete loss (80-90%) of biological activity was admitted. Furthermore, none of the properties of the allegedly pure compounds were described.
The use of high performance liquid chromatography for purification of proteins is generally known in the art.