In this study, the optimization of two-stage fed-batch culture for the production of a foreign protein using recombinant Saccharomyces cerevisiae containing the glucose repressible ADH2/GAP (alcohol dehydrogenase2/glyceraldehyde 3-phosphate dehydrogenase) hybrid promoter was carried out. In the first stage of cell growth on glucose, constant, exponential and ethanol-concentration-control feeding strategies were employed. In the second stage of protein production, ethanol was supplied as the carbon source, and the promoter was activated automatically upon the depletion of glucose. The ethanol concentration was kept lower a certain level to minimize its inhibitory effects and thus to maximize gene expression. With an exponential feeding at the specific growth rate higher than $0.10hr^{-1}$ in the first stage, ethanol was produced intensively, and no high-level production of protein could not be achieved in the second stage. When constant feeding strategy was used in the first stage and ethanol was fed intermittently in the second stage to keep its concentration lower than 10g/L, the final concentration of protein was as high as 286mg/L.
