When I express proteins in bacteria I put at least two stop codons at the end of the gene to increase the termination efficiency. Is this the case in eukaryotic cells too? If I put a single stop codon is there a risk for the ribosomal complex to readthrough and continue translation?
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$\begingroup$ There is no such practice as far as I know. Personally I have never done that and things work fine. You can add a polyadenylation signal, though. Is this a synthetic gene or something ? $\endgroup$– WYSIWYGApr 10, 2013 at 4:06
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1$\begingroup$ From what I understand the eRFs are not perfect but they still work better than RF1 and RF2 which allow for a non trivial amount of readthrough. $\endgroup$– bobthejoeApr 10, 2013 at 5:09
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$\begingroup$ This is a synthetic gene. I already designed a primer with 3 stop codons at the end of the gene. I will post here again if I see any improvement in gene expression (with compared to other similar constructs in our lab). $\endgroup$– Engin YapiciApr 10, 2013 at 19:28
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There is a paper for transfection of mammalian cells here which has a bit of comparison of stop codon and protein yield.
Otherwise this article suggests having a 'rare' codon after the stop to prevent readthrough. But yes, I'm not sure why one wouldn't simply put two stops after each other.
