We want to express more than one protein in yeast. We want to try a one plasmid or two plasmid system. Does anybody know anything about the two plasmid system?
Often plasmids used in yeasts (S. cerevisiae) are so called shuttle vectors that you can use in bacteria or yeast. Using different markers (LEU2, URA3, HIS3, etc.) you can transform yeasts with several different plasmids (best one after the other).
Next to a marker, these plasmids contain an "ARS" (autonomously replicating sequence) and often a "CEN" (yeast centromere); they replicate like chromosomes and their copy number is regulated (on average 1 plasmid per yeast cell).
You can also use derivatives of yeast 2 µm plasmids, which lead to higher plasmid per cell number or even integrative vectors to insert your protein-of-interest genomically.
For high amount protein expression, I can highly recommend the yeast Pichia pastoris. This methanogenic yeast can yield a very high protein expression (protein-of-interest is genomically integrated and under Aox1 promoter; using methanol as C-source can result in up to 30% of overall protein in the cell).
I hope, I could help?
Greetings, Dani
An alternative is the use of one of the four pESC vectors (there are four differents markers: URA, HIS, TRP, LEU) which allow the cloning of two genes under the control of two regulatable GAL promoters on the same plasmid. Then you can easily perform coexpression in yeast without thinking about plasmid stability/compatibility... As the genes are under the control of similar promoters, you can anticipate similar levels of the two proteins. These are commercial plasmids from Agilent Technologies (for more infos : http://www.genomics.agilent.com/article.jsp?pageId=596&_requestid=235541). I personnaly use these system for coIPs and complementation in S. cerevisiae. Hope it helps in some ways...
thanks for the reply
I want to use CEN/ARS system to keep no more than one copy within each yeast cell. If I use two promoter to control expression of two differrent proteins, does the size of the plasmid affect the expression or the transformation efficiency?
I don't think the size of the plasmid has an effect on expression as long as the insert isn't too big...
As transformation efficiency is widely expressed as the number of clones obtained per µg of DNA, the size does matter (1µg contains less copies for larger plasmid..).
ARS plasmids tend to be less stable than CEN (CEN sequence ensures the plasmid segregates as a chromosome during mitosis)
The size of your plasmid should not have a great effect on both expression or transformation efficiency. We take at least 100 ng plasmid DNA to have enough yeast cells transformed (using the simple LiAc method).
I will try ARS/CEN in one plasmid to express two protein under control of two promoters.
Is it true that the linear plasmid could be transfomed more effecttivly than the circular one. I want to make a library. The transformation efficiency is very important.
There are protocols specially designed for high efficiency transformation of more then one plasmid. There is usually an extra step of growing the cells out in rich media for 2 hours before plating on the selective plates. People usually use high efficiency transformation with the two hybrid system.
Cheers,
Evelyn
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