I am modifying the yeast expression vector pYES2 to express a plant transporter. The gene is cloned but I failed to insert an appropriate signal sequence. If the recombinant protein contains multiple transmembrane domains, I am led to believe that these will self assemble upon localization/translation in the ER-membrane. If this is correct, I should be able to use a secretion signal preceding my transporter gene.
There are not any commercial expression vectors which are designed for membrane proteins. I would expect that such a vector feature, the inclusion of a membrane signal sequence, would be available. However the secretion signal, alpha-mating factor sequence, is indeed quite popular for expression in yeast. Perhaps I am looking in the wrong direction for studies on membrane localization in yeast? I cannot find anything on inserting "membrane signal sequences", specifically for yeast.
Hi there,
If you protein localizes at the plasma membrane then you shouldn't have to introduce a targeting sequence as it is already encoded by your GOI and the signal will be efficiently processed by yeast cell machinery. You should worry a bit more about the sequence/codon optimization for getting a proper expression in yeast... As higher eukaryotes have a rather high GC content when compared to yeast... The following should help you: https://academic.oup.com/femsyr/article/22/1/foac047/6731646
Hi James,
which type of protein are you talking about? Which plant species, is it a plasma membrane protein. According to predictions: Does it have multiple transmembrane domains, does it have a signal sequencem is the signal cleavable or not?
There are 100s of examples where plant plasma transporters have been functionally expressed in S. cerevisiae. Rather than going for inducible expression systems I would prefer continuous expression from promotors with medium strength. Such as the pVT100- series of plasmids. In my scientific past codon optimisation has never been necessary.
Best, Jürgen
Thank you Dr. Liger and Dr. Stolz.
For anyone interested, I've found the site below to be helpful in visuallizing transmembrane domains.
https://dtu.biolib.com/DeepTMHMM
I'm still in the troubleshooting phase of this issue. I can see there are multiple transmembrane domains in my protein, although I don't know if any of these can be considered a signal sequence or not. Never mind whether or not it is cleavable...
I figured expression in yeast would be appropriate as a previous study injected capped-RNA of the coding sequence into frog eggs and produced functional transporters. I just need to re-sequence my expression vector maybe..
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