Glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter provides a Strong constitutive expression of heterologous proteins at a level comparable to that seen with the methanol-induced alcohol oxidase (AOXI) promoter in Pichia pastoris. In the present study, strain GS115/G was created to constitutively express secreted human granulocyte-macrophage colony-stimulating factor (hGM-CSF) using GAP-based expression system. With addition of 1% casamino acids, the cultivation of GS115/G resulted in ca. 20% higher cells concentration and ca. 30% higher level of secreted protein concentration in YPD medium, The secreted proteins consisted of an unglycosylated and glycosylated recombinant hGM-CSF with different extent of glycosylation. Instead of using GAP promoter alone in strain GS115/G, the strain GS115/GA that could constitutively and inducibly express recombinant hGM-CSF was generated by transforming host strain GS115/G with AOXI promoter-controlled hGM-CSF expression cassette. In the presence of methanol, the strain GS115/GA expressed recombinant hGM-CSF at a level about two-fold higher than that constitutively expressed by GS115/G. (C) 2003 Elsevier Inc. All rights reserved.