The cell-free method is suitable for rapid and economical production of therapeutic proteins, since it is an open system, which allows us to control the reaction microenvironment to promote folding, solubility of proteins and maximize the protein yield. Consensus interferon is a newly developed type I interferon, a rapid-acting version of interferon that appears more potent than the currently approved pegylated version. Our work aimed to synthesize human consensus interferon-alpha (cIFN-alpha) in cell-free protein expression system of Escherichia colt cells origin. The cloned cIFN-alpha gene in pET101/D-TOPO expression system was used in cell-free IFN production. The system was tested by using a standard construct, GFP (green fluorescent protein) gene was cloned into pIVEX2.3 vector; this gene and our gene, both are under the T7 promoter transcriptional control. The synthesis of active cIFN-alpha gradually increased from 2 to 6 h of the reaction, also reducing the temperature of incubation to <= 30 degrees C maximized its solubility. After purification on nickel-nitrilotriacetate acid (Ni-NTA) resin, the yield of cIFN-alpha was 400 mu g/ml cell-free reaction solution. The resultant cIFN-alpha was fully biologically active as demonstrated by its anti-cancer effect and immunoassay signals. (C) 2011 Elsevier Inc. All rights reserved.