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that allowed for optimal expression in both bacterial and yeast systems [23]. In addition, restriction sites
were incorporated at intervals throughout the length of the sequence to allow for cassette mutagenesis.
Using the Pichia pastoris expression system, 50 mg of purified IFN-τ were obtained from a one-liter
culture.
C. IFN-τ Binding and Signal Transduction
Detailed receptor-binding studies were performed comparing recombinant human IFN-α and IFN-τ
[24]. The K d of 125I-IFN-τ and 125I-IFN-αA for receptor on MDBK cells was 3.9 × 10 -10 M and 4.45 ×
10 -11 M, respectively. Consistent with the higher binding affinity, IFN-αA was several fold more
effective than IFN-τ as a competitive inhibitor. Functionally, the two IFNs had similar specific antiviral
activities, but IFN-τ was 30 fold less toxic to MDBK cells at high concentrations. Phosphorylation of the
signal transduction proteins, Tyk2, Stat1a, and Stat2 did not appear to be involved in the cellular toxicity
associated with IFN-α relative to IFN-τ. Excess IFN-τ did not block the cytotoxicity of IFN-αA,
suggesting that they recognize the receptor differently. While maximal IFN antiviral activity required
only fractional receptor occupancy, toxicity was associated with maximal occupancy. Thus, “spare”
receptors may exist with respect to certain biological properties, and IFNs may induce a concentrationdependent
selective association of receptor subunits.
D. Structural Biology of IFN-τ
In order to better interpret the information derived from the above studies, an understanding of the 3-D
structure of IFN-τ is required. Prior to resolution of the crystal structure, modeling techniques were
employed for structural predictions [10]. For IFN-τ, the homology it shares with the other IFNs can be
exploited. Since the x-ray coordinates for IFN-β are known [2] (Figure 3), it was used as a template for
predicting the topology of IFN-τ. When the sequences of IFN-τ and IFN-β are aligned, the overall
homology is approximately 30%. When residues are compared on the basis of conservative
substitutions, the similarity rises to about 50%, and if only the location of hydrophobic residues is
compared, the similarity is approximately 75%. This is important because hydrophobicity is thought to
be a critical factor in driving protein folding. The interferons IFN-β, IFN-α-2, and several other
cytokines including IL-2, IL-4, growth hormone, and GM-CSF belong to a family in which all share a
four-helix bundle structural motif. Four-helix bundles exhibit a characteristic apolar periodicity in the α
helices where every third or fourth residue is apolar, forming a hydrophobic strip down one side of the
helix, which facilitates packing. The aligned helical regions of IFN-τ show the same apolar periodicity,
suggesting a four-helix bundle motif.
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