Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry.
This paper describes the establishment of flow cytometric methods for recombinant Pichia pastoris strains, and their application to a lab scale fed batch fermentation. Using a strain which secretes human trypsinogen, the viability and the product which remained associated to the cell were measured with propidium iodide and immunofluorescent staining, respectively. Viability decreases significantly below 70% during the methanol fed batch phase, indicating a stress situation triggered by the fermentation conditions. Cell associated product is accumulated earlier after methanol induction than secreted product. These data demonstrate that flow cytometry is a powerful tool for the analysis and optimization of recombinant protein production processes, and they indicate the need to further improve a widely used fermentation protocol for P. pastoris.
Institute of Applied Microbiology, BOKU--University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Hubertus
H
Hohenblum
Nicole
N
Borth
Diethard
D
Mattanovich
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002470:Cell Survival; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D000432:Methanol / Q000378:metabolism*; D010843:Pichia / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development*; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014362:Trypsinogen / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D014362:Trypsinogen; D000432:Methanol