Cytokines, such as interferon-gamma have been shown to have adjuvant and growth promoting activity in poultry and livestock and have the potential to be used as alternatives to antibiotics. We have developed an efficient system for commercial-scale synthesis of recombinant chicken interferon-gamma (ChIFN-gamma) using Brevibacillus choshinensis as the host for protein production. The ChIFN-gamma expression vector, pNCIFN, was constructed using the novel Escherichia coli-B. choshinensis shuttle vector, pNCMO2. ChIFN-gamma expression was optimized by investigating different culture conditions and different host B. choshinensis mutants. The highest level of production was observed using the B. choshinensis HPD31-MB2 strain grown at 30 degreesC, where ChIFN-gamma was produced at approximately 300-500 mg/ L. ChIFN-gamma was also produced as a His-tagged fusion protein by using the pNCHis-IFN expression vector, a derivative of pNCMO2. The protein was constitutively secreted into the culture supernatant and could be partially purified in a single step using a Ni-nitrilotriacetic acid column. This recombinant His-ChIFN-gamma was shown to have the same biological activity as native ChIFN-gamma.