title: Carboxy-terminal truncated rhuIFN-gamma with a substitution of Gln133 or Ser132 to leucine leads to higher biological activity than in the wild type.
abstract: The biological function of the 20 C-terminal amino acids of human interferon-gamma (IFN-gamma) was examined by recombinant DNA methodology. Six truncated IFN-gamma analogues were produced by modification of the 3' end of the coding sequence of the cloned gene, insertion into a vector with the trc promoter and expression of the recombinant IFN-gamma analogue genes in Escherichia coli strain JM 105. The IFN-gamma analogue proteins were shortened by 10 (C-10L), 11 (C-11, C-11L), 14 (C-14L), 19 (C-19L) and 20 (C-20) amino acid. Four of these constructs were modified to have a C-terminal leucine. The expression rates of precipitating IFN-gamma variants in E. coli cells (wild type, C-10L, C-11, C-11L) amount to 35-40% of the total protein, in contrast to 14-21% for the mainly soluble ones (C-14L, C-20). The variant C-19L has an exceptional position in its solubility behaviour with a nearly 1:1 distribution between its soluble and insoluble form by an expression rate of 8%. The purification protocol of the insoluble variants contains a denaturing and a renaturation step. The characteristic step for purification soluble IFN-gamma is HPLC cation-exchange chromatography. The antiviral activities of the variants lacking 14 or more amino acids are less than 2% of the wild-type activity. The variants C-10L, C-11 and C-11L show higher biological activities than wild-type IFN-gamma. The most active variant, C-10L, with leucine as the last C-terminal amino acid, has a fourfold higher specific antiviral activity (A549 cells, encephalomyocarditis virus). Removal, but not replacement of the leucine, represented by the variant C-11, reduces the biological activity compared with variant C-10L. The activity of C-11 is, nevertheless, higher than in the wild type. Comparing the secondary structures, as judged by CD analyses, no significant differences for C-10L, C-14L and C-20, compared with wild type, are observed. Also, all molecules, including the wild-type protein, exist as dimers under physiological conditions. There is a correlation between the grade of truncation and the pI values, which range from pI = 10.4 (wild type) to pI = 8.0 (C-20). The variant C-10L demonstrates a higher temperature stability (tm = 55 degrees C) compared with wild type (tm = 53 degrees C). Perhaps this higher stability will result in a longer half-life in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Fraunhofer Institut für Toxikologie und Molekularbiologie, Abteilung Gentechnologie, Hannover, Federal Republic of Germany.
authors.forename: O
authors.initials: O
authors.lastname: Slodowski
authors.forename: J
authors.initials: J
authors.lastname: Böhm
authors.forename: B
authors.initials: B
authors.lastname: Schöne
authors.forename: B
authors.initials: B
authors.lastname: Otto
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000187:drug effects; D014644:Genetic Variation; D005973:Glutamine; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D007930:Leucine; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins; D012694:Serine
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005973:Glutamine; D012694:Serine; D007371:Interferon-gamma; D007930:Leucine