Carboxy‐terminal truncated rhuIFN‐γ 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)