Stable continuous constitutive expression of a heterologous protein in Saccharomyces cerevisiae without selection pressure

Autor: Jochen Kuhla, Martin Küenzi, Michael Ibba, Alan Smith
Rok vydání: 1993
Předmět:
Zdroj: Applied Microbiology and Biotechnology. 39:526-531
ISSN: 1432-0614
0175-7598
DOI: 10.1007/bf00205045
Popis: The stability of heterologous protein expression in Saccharomyces cerevisiae during continuous culture without selection for plasmid-containing cells was investigated. The protein chosen was the leech thrombin inhibitor desulphato-hirudin, which is tolerated well by S. cerevisiae when over-expressed. Expression was from a 2-mu derived multicopy vector containing a synthetic hirudin gene under control of the constitutive glyceraldehyde-3-phosphate dehydrogenase derived GAPFL promoter. The behaviour of the system was studied at three dilution rates (D) corresponding to approximately 30% (0.06 h-1), 60% (0.12 h-1) and 90% (0.17 h-1) of the estimated maximum D. The level of plasmid loss was low at all Ds, with only 5-10% plasmid-free cells observed at 75 generations. The plasmid was most stably maintained at the intermediate D of 0.12 h-1, where the rate of loss was comparable to the loss of the native 2-mu plasmid. Hirudin expression was also highest at D = 0.12 h-1, possibly as a result of cell lysis at D = 0.06 h-1 and D = 0.17 h-1, leading to the release of vacuolar proteases and subsequent proteolysis of hirudin. Differences in expression levels were not a result of changes in plasmid copy number, which was in the range 40-60 throughout all three experiments. The high stability of this system at all Ds investigated shows that heterologous protein expression is not a burden to S. cerevisiae when the protein expressed is tolerated well.
Databáze: OpenAIRE
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