Induction and measurement of UPR and osmotic stress in the yeast Pichia pastoris.
Unfolded protein response (UPR) is a major reaction to intrinsic stress of eukaryotic organisms and is also related to environmental stress reactions. Among yeasts, stress regulation has mainly been investigated in Saccharomyces cerevisiae, while other species with biotechnological or medical interest are less well understood. Pichia pastoris as one example has emerged as a favorite production platform for recombinant proteins during the last two decades. UPR and environmental stress are well known to interfere with the production of recombinant proteins as well as other technologically relevant processes, so that the demand for well-documented protocols to measure such stress reactions has strongly increased. Here, we describe protocols for the induction of UPR and osmotic stress, as well as for the quantitative measurement of cellular stress reactions at the levels of transcripts, proteins, and metabolites. As such protocols need to be adapted for a new species of interest, the guidelines presented here should enable researchers to study P. pastoris directly without the hassle to modify standard protocols designed for the model organism S. cerevisiae first.
Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
Martin
M
Dragosits
Diethard
D
Mattanovich
Brigitte
B
Gasser
D050976:Basic-Leucine Zipper Transcription Factors / Q000096:biosynthesis; D004229:Dithiothreitol / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics*; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis; D014415:Tunicamycin / Q000494:pharmacology; D056811:Unfolded Protein Response / Q000235:genetics
D050976:Basic-Leucine Zipper Transcription Factors; D005656:Fungal Proteins; C091341:HAC1 protein, S cerevisiae; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014415:Tunicamycin; D004229:Dithiothreitol