Carboxy‐terminal truncated rhuIFN‐γ with a substitution of Gln133 or Ser132 to leucine leads to higher biological activity than in the wild type

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Abstract
The biological function of the 20 C‐terminal amino acids of human interferon‐γ (IFN‐γ) was examined by recombinant DNA methodology. Six truncated IFN‐γ 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‐γ analogue genes in Escherichia coli strain JM 105. The IFN‐γ 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‐γ 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‐γ 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‐γ. 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°C) compared with wild type (tm= 53°C). Perhaps this higher stability will result in a longer half‐life in vivo. This investigation points out the importance of the basic amino acids cluster Lys128–Arg131. Partial or complete removal of this region leads to a drastic loss in biological activity, while truncations near to this region result in a significant increase in activity.