Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni2+-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being similar to210 and 75 mg/L, respectively. The rhIFNs expressed within this system were biologically active (similar to1,1 x 10(8) IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest. (C) 2004 Elsevier Inc. All rights reserved.