Overexpression of rhIFN-alpha 2b was obtained by synthesizing a codon optimized gene for IFN-alpha 2b and expressing it in the form of inclusion bodies (1Bs) in Escherichia coli. The recombinant plasmid pRSET-IFN alpha, which had the IFN-alpha 2b gene under the T7 promoter, was coexpressed with plasmid pGPI-2, which carried the gene for T7 RNA polymerase under the heat inducible lambda P-L promoter. This two plasmid expression system was optimized with respect to heat shock time, media, and time of induction in shake flask cultures. This was then scaled up into a bioreactor to get a maximum volumetric product yield of 5.2 g/L at a final OD600 of 67. At this point, the IBs represented similar to 40 % of the total cellular protein. This high specific product yields eased the further downstream processing steps and improved product recoveries. The IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of similar to 3 g/L, which is maximum reported in the literature. The bioassay of the refolded protein gave a specific activity of similar to 3 x 109 IU/mg protein. (c) 2005 Elsevier Inc. All rights reserved.