Abstract
BackgroundBaker’s yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins. For the manufacture of heterologous proteins with activities deleterious to the host it can be desirable to minimise production during the growth phase and induce production late in the exponential phase. Protein expression by regulated promoter systems offers the possibility of improving productivity in this way by separating the recombinant protein production phase from the yeast growth phase. Commonly used inducible promoters do not always offer convenient solutions for industrial scale biopharmaceutical production with engineered yeast systems.ResultsHere we show improved secretion of the antimicrobial protein, human β-defensin-2, (hBD2), using the S. cerevisiae MET17 promoter by repressing expression during the growth phase. In shake flask culture, a higher final concentration of human β-defensin-2 was obtained using the repressible MET17 promoter system than when using the strong constitutive promoter from proteinase B (PRB1) in a yeast strain developed for high-level commercial production of recombinant proteins. Furthermore, this was achieved in under half the time using the MET17 promoter compared to the PRB1 promoter. Cell density, plasmid copy-number, transcript level and protein concentration in the culture supernatant were used to study the effects of different initial methionine concentrations in the culture media for the production of human β-defensin-2 secreted from S. cerevisiae.ConclusionsThe repressible S. cerevisiae MET17 promoter was more efficient than a strong constitutive promoter for the production of human β-defensin-2 from S. cerevisiae in small-scale culture and offers advantages for the commercial production of this and other heterologous proteins which are deleterious to the host organism. Furthermore, the MET17 promoter activity can be modulated by methionine alone, which has a safety profile applicable to biopharmaceutical manufacturing.
Highlights
Baker’s yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins
High‐level expression of human β‐defensin‐2 from the MET17 promoter is induced by methionine depletion To determine if the repressible MET17 promoter was more efficient for human β-defensin-2 (hBD2) expression than the strong constitutive PRB1 promoter, expression plasmids were produced harbouring the gene for hBD2 downstream of either the MET17 or PRB1 promoter
The ultra-performance liquid chromatography mass spectrometry (UPLC-MS) data indicated that a higher hBD2 concentration was obtained in DYB7 using the MET17 promoter repressed by 1000 μM methionine compared to the PRB1 promoter in less than half the incubation time (Fig. 2a, b)
Summary
Baker’s yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins. Regulation of these promoters may interfere with the cellular metabolism and in many cases the regulation is not tight enough to completely shut off transcription. This issue was addressed by use of the tetracycline (Tet-On/Off ) promoters, which are either inducible or repressible [4]. Gene expression is activated as a result of binding of the Tet-Off or Tet-On protein to an element located within an inducible promoter One advantage of this system is that promoter regulation with the tetracycline derivative, doxycycline does not interfere with the yeast cellular metabolism. Alternative promoter systems with safe and simple regulation are desirable for some large scale biopharmaceutical processes
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