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Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris.

PLoS ONE. 2015; 
Edwards-JonesBryn,AwRochelle,BartonGeraint R,TredwellGregory D,BundyJacob G,LeakDav
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Proteins, Expression, Isolation and Analysis … Blots shown are representative of at least three independent experiments performed 26 Caspase-3 activity assay Caspase-3 activity in the lysates was detected using a colorimetric assay kit (Genscript, Piscataway, NJ, USA) according to the manufacturer's instructions … Get A Quote

摘要

We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analys... More

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