Combined use of GAP and AOX1 promoter to enhance the expression of human granulocyte-macrophage colony-stimulating factor in Pichia pastoris

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 (AOX1) 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 AOX1 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.