Methods for Preparation of Recombinant Cytokine Proteins V. Mutant Analogues of Human Interferon-γ with Higher Stability and Activity

Mutant analogues of recombinant human immune interferon (IFN-γ) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-γ analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-γ was studied by reversed-phase HPLC. IFN-γ and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19°C and four times enhancement of biological activity compared with intact IFN-γ molecule.