Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in <Emphasis Type="Italic">Pichia pastoris</Emphasis> |
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Authors: | C Ruth T Zuellig A Mellitzer R Weis V Looser K Kovar A Glieder |
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Institution: | 1.Institute of Molecular Biotechnology,Graz University of Technology,Graz,Austria;2.Research Centre Applied Biocatalysis,Graz,Austria;3.Institute of Biotechnology,University of Applied Sciences Zurich (ZHAW),Waedenswil,Switzerland;4.VTU Technology Technologieentwicklungsgesellschaft m.b.H.,Graz,Austria |
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Abstract: | Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression
in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the
application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently
regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy.
Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the
applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch
processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the
promoter choice. |
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Keywords: | Pichia pastoris AOX1 promoter variants Porcine trypsinogen Production window |
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