Hello,
I would like to co-express a large (250 kDa) chloride ion channel, along with one of its interacting proteins (50kDa, soluble) in yeast. We have already optimized the expression of the membrane protein in yeast, so I would like to stick with the strain we have been using as this contains a protease deletion which has proved useful in reducing proteolysis of this large protein. The problem is that this particular stain is only ura3 deficient, meaning cloning into another plasmid and using another auxotrophic marker is not an option. The strain is also lys2 deficient so could I use a -ura media with alpha aminoadipate as a nitrogen source as the two selection markers? Or I was thinking of using a bidirectional expression vector such as this pBEVY one.
Has anyone tried to do something similar and would be able to offer any advice?
Many thanks!
Ellie
https://www.addgene.org/51229/
There are plenty of selection markers you can use with yeast. For example there are range of antibiotics you can use as geneticin, bleomycin, zeocin, hygromycin B and so many others that can be used in eukaryotes since there are resistance cassettes that can be incorporated in the plasmid.
Do you mean that your strain is uracil (ura3) and lys2 (lysine) deficient or its leu2 (leucine deficient)? Leucine deficiency is more common in yeast strains. In either case you can use miniaml medium where you dont supplement either the two componentes the strain is auxotrophic to.
Minimal media for yeast contains: 20 g/L of glucose, 1.7 g/L of Yeast nitrogen base without amino acids and without ammonium sulphate and 2.7g/L of ammonium sulphate. If the strain has only those 2 auxotrophies, when transformed with the corresponded vectors it will grow and transformants will be selected.
hi
i think you could cloned your genes Such as the following steps :
1- 250 kd gene product cloned in" pPIC9K " vector and then transfect in p.pastoris SMD1168 (His selection/ AOX1 promoter)
2- after selection the right clones you could 50 kd gene product cloned in pGAPZ series vector ( if you want secretion the product from p.pastoris you must used pGAPZ-alpha series ) ( Zeocin selection / GAP promoter )
https://tools.thermofisher.com/content/sfs/brochures/B-067202_Pichia_Flyer.pdf
good luck
Hi Rui, thanks for your response. Yes the strain is lysine deficient, which does seem to be a less common auxotrophic marker. Most of the examples of the use of this as a selective marker are based on the ability of lys2 deficient strains to utilize alpha-aminoadipate as a nitrogen source, rather than through complementation with a vector? I'm also struggling to find any vectors containing LYS2?? Am I missing something?
Hi Reza, thanks for your response. I'm afraid expression in pichia is no good for our protein.
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