Evidence for a polypeptide segment at the carboxyl terminus of recombinant human gamma interferon involved in expression of biological activity.
A panel of 18 murine monoclonal antibodies was raised in BALB/c mice to the full-length, 146 amino acid residue recombinant human gamma interferon (rHuIFN gamma-A). Two monoclonal antibodies, designated 47N3-6 and 30N47-1, were purified from ascites tumors and further characterized. Antibody 47N3-6 neutralized both the antiviral and antiproliferative activities of rHuIFN gamma-A. Both Western blotting and enzyme-linked immunosorbent assays indicated that antibody 47N3-6 could bind to rHuIFN gamma-A as well as to a genetically engineered truncated form lacking the first three amino-terminal residues (rHuIFN gamma-D) but did not recognize a genetically engineered variant terminating at residue 131 (rHuIFN gamma-B). This antibody also demonstrated binding to a 15 amino acid residue oligopeptide, designated F-1, corresponding to residues 132-146 at the carboxyl terminus of rHuIFN gamma-A. Chemical cleavage of peptide F-1 with cyanogen bromide produced two fragments that were separated by reversed-phase high-pressure liquid chromatography. Dot-blot analysis indicated that antibody 47N3-6 could bind to a fragment, KRKRSQHse, derived from residues 132-137 of rHuIFN gamma-A, but could bind only weakly to the cyanogen bromide fragment corresponding to residues 138-146. It was consistent with these results that antibody 47N3-6 demonstrated binding to a form lacking the five carboxyl-terminal amino acids (rHuIFN gamma-D') but did not bind to a synthetic polypeptide corresponding to residues 138-146. Peptide F-1 exhibited neither antiviral nor antiproliferative activity, and it did not antagonize the antiviral activity of rHuIFN gamma-A.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D010446:Peptide Fragments / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007371:Interferon-gamma