Transition phase in the production of recombinant proteins in yeast under the ADH2 promoter: an important step for reproducible manufacturing of a malaria transmission blocking vaccine candidate |
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Authors: | S B Noronha D C Kaslow J Shiloach |
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Institution: | (1) Biotechnology Unit, LCDB, NIDDK, NIH, Bethesda, MD 20892, USA, US;(2) Molecular Vaccine Section, NIAID, NIH, Bethesda, MD 20892, USA, US |
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Abstract: | TBV25H, a malaria transmission blocking vaccine candidate, has been cloned in Saccharomyces cerevisiae under the control of the glucose repressed ADH2 promoter. Available fermentation procedures for production of this protein have been unsatisfactory, mainly because of irreproducibility.
This work presents an efficient and reproducible method for the production of this vaccine candidate by implementing a three-stage
fermentation process. During the first (glucose fed-batch) phase, the promoter is repressed and the culture is allowed to
grow exponentially. In the second stage, the glucose supply is provided at a slower constant rate. In the third (ethanol consumption)
stage, accumulated ethanol is first allowed to be consumed and an external ethanol supplement is then added as required. The
promoter is fully derepressed in this phase, and TBV25H is synthesized. The period of glucose limitation was concluded to
be essential for reproducibility. It is presumed that during this period, the culture moves gradually from glucose to ethanol
utilization, derepressing the promoter, activating recombinant protein biosynthesis and consequently resuming metabolism without
the typical diauxic phase of batch cultures.
Received 21 October 1997/ Accepted in revised form 15 January 1998 |
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Keywords: | : ADH2 TBV25H Saccharomyces malaria |
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