Pichia pastoris strains with capability to sequentially express two recombinant proteins under control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GAP) and the methanol-induced alcohol oxidase (AOX1) promoters have been constructed. The intracellular beta-galactosidase (beta-gal), secreted human serum albumin (hSA) and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) are used as model proteins to demonstrate their sequential expression and separate recovery. The strain X33/ZCSF cultivated in YPD medium results in high-level constitutive expression of p-galactosidase activity and methanol-induced, secreted highly glycosylated hGM-CSF expression. The p-galactosidase activity and hGM-CSF are separately recovered from cells pellet and supernatant. On the other hand, the strain GS115/CSFA cultivated in YPD medium constitutively expresses secreted hGM-CSF along with cell growth. After a high cell concentration is achieved, the YPD medium is then replaced with YPM medium to induce the expression of the AOX1 promoter controlled hSA. The sequentially expressed hGM-CSF and hSA can be separately recovered from the spent YPD and YPM medium. (C) 2003 Elsevier Science B.V. All rights reserved.
