Sequential expression of recombinant proteins and their separate recovery from a Pichia pastoris cultivation

✍ Scribed by Jyh-Ming Wu; Lieng-Lung Chieng; Tsu-An Hsu; Cheng-Kang Lee

• Book ID: 104419890
• Publisher: Elsevier Science
• Year: 2003
• Tongue: English
• Weight: 391 KB
• Volume: 16
• Category: Article
• ISSN: 1369-703X
• DOI: 10.1016/s1369-703x(03)00017-2

✦ Synopsis

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 ␤-galactosidase (␤-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 ␤-galactosidase activity and methanol-induced, secreted highly glycosylated hGM-CSF expression. The ␤-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.