Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena.
Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.
Institute of Molecular Biotechnology and Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria.
Roland
R
Weis
Ruud
R
Luiten
Wolfgang
W
Skranc
Helmut
H
Schwab
Marcel
M
Wubbolts
Anton
A
Glieder
D017209:Apoptosis / Q000502:physiology*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D005947:Glucose / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D016254:Mutagenesis, Insertional; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic / Q000502:physiology
D004271:DNA, Fungal; D011994:Recombinant Proteins; D005947:Glucose