Stable continuous constitutive expression of a heterologous protein in Saccharomyces cerevisiae without selection pressure.
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.
Ibba
M
M
Department of Biotechnology, Ciba AG, Basel, Switzerland.
Kuhla
J
J
Smith
A
A
Küenzi
M
M
D000818:Animals; D001709:Biotechnology; D015870:Gene Expression; D005822:Genetic Vectors; D006629:Hirudins / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D007865:Leeches / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D006629:Hirudins; D011994:Recombinant Proteins; C060225:desirudin