Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1986-02-04
1989-05-09
Hazel, Blondel
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
424 855, 530351, 435811, C12P 2100, C07K 1526, A61K 4502
Patent
active
048289892
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD OF THE INVENTION
This invention relates to a process for purifying a protein. More particularly, the invention relates to a process for purifying a protein that forms highly insoluble aggregates during the growth of cells transformed with DNA sequences coding for the protein, the protein being solubilizable and renaturable from the extracts of those cells to produce a conformation that is characterized by less than three surface cysteine amino acid residues.
A preferred protein purified according to the process of this invention is immune or gamma interferon (IFN-.gamma.). The purified, homogeneous and stable gamma inteferon produced according to the invention may be utilized in the therapeutic treatment of viral infections, tumors or cancer as well as in immunomodulation applications and methods.
BACKGROUND OF THE INVENTION
Purification procedures such a precipitation, molecular sieve chromatography, electrophoresis, affinity chromatography and covalent chromatography are well known in the art and have been utilized in the purification of proteins from cell extracts. However, purification of proteins produced by cells transformed by recombinant DNA sequences that code for them has posed unique and difficult problems.
Preferably, the level of expression of the recombinant DNA sequence is high and therefore the host cell transformed by that DNA sequence produces a large amount of the desired protein within the cell. Accordingly, the tranformed cell accumulates large numbers of foreign protein molecules. These molecules may then interact with each other to form highly insoluble aggregates, not typically found in the normal cell. The host cell then responds to this unusual accumulation of foreign protein by forming inclusion bodies composed of the foreign protein aggregates. Purification of these foreign proteins in a biologically active form, therefore, requires a means of solubilizing these highly insoluble protein aggregates in such a way to preserve or to enable recovery of their native conformation and a means for purifying the soluble protein in a manner that maintains the biological activity of the protein.
A genetically engineered protein of great value to the health field is interferon. ln this application we will use the interferon nomenclature announced in Nature, 286, p. 110 (Jul. 10, 1980). "IFN" will designate interferon, "IFN-.alpha." will designate leukocyte interferon, "IFN-.beta." will designate fibroblast interferon, and "IFN-.gamma." will designate gamma interferon.
IFN is a cellular protein displaying antiviral activity against a broad range of viruses through induction of cellular RNA and protein synthesis directed against virus replication. For example, human IFN has been used to combat the viral activity of the following: respiratory infections; [Texas Reports on Biology and Medicine, Vol. 35, pp. 486-96 (1977) (hereinafter referred to as Texas Reports)]; herpes simplex keratitis [Texas Reports, pp. 497-500; R. Sundmacher, "Exogenous Interferon In Eye Diseases", International Virology IV, The Hague, Abstract nr. w2/11, p. 99 (1978)]; acute hemorrhagic conjunctivitis [Texas Reports, pp. 501-510]; adenovirus keraton conjunctivitis [A. Romano et al., ISM MemoI-A8131 (October, 1979)]; varicella-zoster [Texas Reports, pp. 511-515]; cytomegalovirus infection [Texas Reports, pp. 523-527]; and hepatitis B [Texas Reports, pp. 516-522]. See also W. E. Stewart, II, The Interferon System, pp. 307-321 Springer-Verlag (2 ed.) (1981) (hereinafter referred to as The Interferon System).
IFN has other effects in addition to its antiviral action. For example, it antagonizes the effect of colony stimulating factor, inhibits the growth of hemopoietic colony-forming cells and interferes with the normal differentiation of granulocyte and macrophage precursors [Texas Reports, pp. 343-349]. It also inhibits erythroid differentiation in DMSO-treated Friend leukemia cells [Texas Reports, pp. 420-428].
IFN may also play a role in the regulation of the immune response. For example, depending up
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patent: 4405601 (1983-09-01), McEntire et al.
patent: 4518526 (1985-05-01), Olson
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Chemical Abstracts, vol. 92, Abstract No. 74138x, 1980.
Ducommun Garance M.
Prior Chrisopher P.
Haley, Jr. James F.
Hazel Blondel
Yankwich Leon R.
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