Longer Explanation: I would like to express a fusion of protein X and Y in prokaryotes. However, I want Y to be expressed by itself as well (from the same promoter). In a mammalian system (which I am more familiar with) I would just simply put a read-through IRES in between X and Y. This would result in the gene products XY as well as Y alone (ratio depending on how good the used IRES is). An alternative would be to put the FMDV 2A translational skip peptide in between X and Y which would yield the gene products XY and X + Y (ratio depending on the efficiency of 2A). However, I don't know of any IRES that can be used in prokaryotes and also the FMDV 2A peptide is not active in prokaryotes. I was also thinking if a protease site in between X and Y would work (if it is only partially efficient). Also, one could consider a read-through stop-codon (amber) but that would result in XY and X rather than XY and Y (the fusion must be XY and not YX). Anyway, if any of you good prokaryote scientists out there have any suggestions for an eukaryotic scientist, please help! Thanks!!
Daniel, you do not need an IRES sequence in prokaryotes because they naturally will translate multiple reading reading frames from the same transcript. So you can have a single promoter transcribe multiple genes
Promoter A--rbs--OrfA--rbs--OrfB--rbs--OrfC etc
So what Martin writes above is correct except that you don't need the second promoter A if the two open reading frames are adjacent to each other (but it doesn't hurt to do it like he drew).
Thanks for the advice! I think I'll go with something like:
Promoter-rbs-OrfX-rbs-OrfY-stopcodon
By avoiding a stopcodon after OrfX and making sure I have a readthrough rbs in between the Orfs this should hopefully give me the products XY and Y from the same RNA which is my goal.
After reading some more I think I'll go with a partly suboptimal SD-sequence for the first rbs and an optimal SD for the second rbs. I am striving for XY:Y in a ratio of 1:5. Let's see if it works out! Thanks for you valuable help!!
Daniel - you must INCLUDE a stop codon after OrfX and OrfY otherwise translation will continue and you make incorrect proteins. What you mean is that you should avoid having a transcriptional stop after OrfX. But you MUST have a translational stop codon.
Also, if you want to make more of Y than of X, I would position it first with a strong RBS and then the reduced expression OrfX second with a weaker RBS.
Hi again Michael! Thanks for your advice! However, I am not trying to produce protein X and protein Y. I want to produce the protein Y and the fusion protein XY. Kind of unusual I guess, but it suits my purposes. Hence I want some translation to start from the startcodon of X, readthrough all the way to the stopcodon of Y thereby expressing the fusion XY. From the samt RNA I want to produce only Y, hence adding a RBS just before Y (in the middle of the RNA) would hopefully do the trick, just like you suggested! Thanks again!
Hi Daniel,
How was your experience with prokaryotic RBS in terms of expression for the second transgene? What about the fused protein?
Thanks.
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