Structure-function studies of interferon-α based on random mutagenesis and expression in vitro

An efficient procedure for random chemical mutagenesis was used to create analogs of human interferon (IFN)-α4. Unique restriction enzyme sites were introduced into the human IFN-α4 gene to enable cassetting of the gene for localized random mutagenesis. Single-stranded IFN-α4 DNA was treated with nitrous acid, followed by second-strand synthesis using reverse transcriptase. A 72 base pair cassette spanning the coding region for amino acid residues 120 to 136 (120–136 region) was isolated and cloned into a phagemid vector adjacent to a GC-rich sequence. A DNA segment comprising the IFN-α4 cassette sequence and the GC clamp was excised and electrophoresed on a denaturing gradient gel, which allowed the separation from unmutated DNA of DNA fragments with single base pair changes. DNA fragments with mobility different from that of the unmutated fragment were pooled and cloned into an expression vector. Using this procedure, mutations were found in the DNA of 48% of the clones analyzed. However, mutations at two “hot spots” accounted for 89% of these clones. Four of the IFN-α4 analogs with mutations in the 120–136 region were expressed in vitro. The antiproliferative activities on human Daudi cells of most of the analogs were less than 0.2% of the activity of unmodified IFN-α4, suggesting that the integrity of the carboxy terminus is important for the antiproliferative activity of human IFN-α4.