The effect of hydrostatic pressure on the secondary structure of recombinant human interferon-gamma (rhIFN-gamma) and its biologically inactive truncated form rhIFN-Delta C15 has been studied using Fourier-transform IR (FTIR) spectroscopy. In situ observation of the pressure-induced changes using the diamond anvil cell shows that the alpha-helical structure is mainly transformed into disordered structure at high pressure. Increasing pressure also induces the formation of a gel. Addition of 0.5 M MgCl(2) significantly reduces the pressure stability. Releasing the pressure below 300 MPa results in the formation of intermolecular antiparallel beta-sheets, which is seldom observed. This suggests that the intermolecular beta-sheet of rhIFN-gamma is stabilized by electrostatic interactions that are disrupted at high pressure. For comparison we also studied the effect of temperature. Temperature-induced changes reflect extensive transformation of alpha-helical structure into intermolecular antiparallel beta-sheet, as is usually observed for most proteins.

Recombinant human interferon gamma (IFN-gamma), produced in Escherichia coli, was selectively truncated at its C-terminus with chymotrypsin, clostripain or plasmin. The C-terminal amino acid residues of the three truncated IFN-gamma variants were identified as Phe136, Arg129 and Lys128, indicating the removal of 7, 14 and 15 amino acid residues from the full-length molecule. The absence of seven C-terminal residues did not influence the binding of IFN-gamma to its receptor. In contrast, the truncation of 14 residues resulted in a decrease in the Ka value to 1/24, as determined by surface plasmon resonance analysis. The removal of one additional amino acid residue from the C-terminal region of IFN-gamma led to a marked loss of receptor-binding capacity and biological activity. These observations demonstrate that Arg129 is an essential part of a functionally important C-terminal IFN-gamma sequence that is involved in receptor interaction.

D9D10, a monoclonal antibody that inhibits the biological activity of human interferon-g (IFN-g), was used to generate monoclonal anti-idiotypic antibodies. After a first selection, the monoclonal anti-idiotypic antibody AA1E5 was chosen to be fully characterized. To the best of our knowledge this is the first description of a monoclonal antibody with an IFN-g-like antiviral activity; AA1E5 competed with IFN-g for binding to D9D10 indicating its anti-idiotypic character. However, AA1E5 also fully mimics HuIFN-g as it not only binds to the HuIFN-g-receptor, where it competes with HuIFN-g, but more importantly AA1E5 and its Fv fragment, cloned and expressed in Escherichia coli, mimic the antiviral activity of HuIFN-g. Indeed, 15 mg of AA1E5 and 2.5 mg of its Fv fragment had an effect comparable to that of 10 IU of HuIFN-g in an antiviral assay on A549 cells. Sequence comparison between the complementarity determination regions of the antibody and the sequence of HuIFN-g revealed that both the heavy chain variable domain, V H , and the kappa light chain variable domain, V k , have epitopes of 3±4 amino acids that are present in the HuIFN-g sequence, some of which contribute to receptor binding, as identified by Walter et al.

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