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Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris.

Ruth, Zuellig, Mellitzer, Weis, Looser, Kovar, Glieder.
Graz University of Technology
Systems and Synthetic Biology 2010, 4(3):181-91
Gene names: trypsinogen. Host systems: Pichia pastoris[Yeast]. Gene species: Porcine (Pig)[Mammalian]. Optimized: Yes.
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.
Comments: Dr Glieder's team in Graz Univ Technol used DNA2.0 to design and synthesize the porcine trypsinogen for expression in Pichia. Several promoters were tested for expression and process optimization.