Human interferon gamma (hIFN-gamma) has an antiviral, immunoregulatory and anti-tumor properties. It is majorly used for the treatment of chronic granulomatous disease and severe malignant osteopetrosis. In the present investigation, cell level strategies were applied to address the expression machinery related problems, which are bottle neck for protein expression in Pichia pastoris. The gene encoding hIFN-gamma was successfully cloned and expressed in P. pastoris (GS115) under the control of AOX promoter. About 200 mu g/L of recombinant human interferon gamma (rhIFN-gamma) was expressed after 1% methanol induction. The expression of rhIFN-gamma was further enhanced by 2.67 fold by co-expressing the gene encoding protein disulphide isomerase (PDI) along with hIFN-gamma. Furthermore, the effect of codon bias was examined by cloning codon optimized hIFN-gamma in P. pastoris (GS115) that resulted in about 9 fold enhancement of rhIFN-gamma production. Optimization of process parameters viz., temperature, pH, methanol concentration, inoculum size and agitation rate enhanced the production of rhIFN-gamma by 12.5 fold. The purification with IMAC (Immobilized metal ion affinity chromatography) showed about 56.5, 63.83 and 80% purity of rhIFN-gamma from GS-hIFN-gamma, GS-hIFN-gamma-PDI and GS-hIFN-y(opt) strains, respectively. (C) 2016 Elsevier Ltd. All rights reserved.