title: Amino terminus is essential to the structural integrity of recombinant human interferon-gamma.
abstract: Species lacking either 8 or 10 residues at the amino terminus of recombinant human interferon-gamma (Hu-IFN-gamma) were generated by limited digestion with Staphylococcus aureus V8 protease. A crude digest, consisting predominantly of these species, were completely inactive in inducing antiviral activity and the expression of HLA-DR antigens on HL-60 cells. The NH2-terminal deletion fragments were separated from residual intact IFN-gamma and from smaller polypeptides by reverse phase high performance liquid chromatography (HPLC) at pH 2.2. Intact IFN-gamma, purified by HPLC and subsequently refolded by dilution in 0.1 M sodium phosphate buffer (pH 7.5, 0.1% bovine serum albumin) was similar to untreated IFN-gamma in terms of binding to its cell surface receptor and in inducing antiviral activity and the expression of HLA-DR molecules. Conversely, biological activity was not detected in purified fragments 8-139 and 10-139. Examination of fragments 8-139 and 10-139 by far-UV circular dichroism revealed that cleavage of 8-10 residues at the amino terminus accompanied a dramatic change in secondary structure (6% alpha-helical and 36% beta-sheet content) as compared to untreated or HPLC-purified IFN-gamma (66% alpha-helix and 0% beta-sheet content). In summary, these results indicate that the amino terminus contributes to the structural integrity of the IFN-gamma molecule.
authors.affiliation: Division of Cytokine Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
authors.forename: H H
authors.initials: HH
authors.lastname: Hogrefe
authors.forename: P
authors.initials: P
authors.lastname: McPhie
authors.forename: J B
authors.initials: JB
authors.lastname: Bekisz
authors.forename: J C
authors.initials: JC
authors.lastname: Enterline
authors.forename: D
authors.initials: D
authors.lastname: Dyer
authors.forename: D S
authors.initials: DS
authors.lastname: Webb
authors.forename: T L
authors.initials: TL
authors.lastname: Gerrard
authors.forename: K C
authors.initials: KC
authors.lastname: Zoon
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006684:HLA-DR Antigens / Q000096:biosynthesis; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology
chemical_list: D006684:HLA-DR Antigens; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007371:Interferon-gamma; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase