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Designing a Plasmid that Will Produce a Fusion Protein


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Hi, I'm reading a couple of papers about making transducible proteins (proteins that can pass through the cell membrane) and it seems the way to do this is to make a fusion by attaching a protein transduction domain (PTD) onto whatever protein your interested in making transducible. Now it seems this is done by cloning the gene(s) needed to make your protein of interest into a plasmid that already contains a PTD. I'm just wondering how the PTD plasmid is made in the first place. It seems like what you have to do is somewhere in between your promoter site and your multiple cloning site you have to insert a 6 histidine sequence, followed by your PTD sequence, and then a glycine residue. After this your multiple cloning site should follow. Now in one of the papers below it says you should delete the 5' UTR sequence from whatever gene your cloning in. However does this mean that the start codon from the gene of interest should also be deleted? It makes sense if that is the case since I could see how that would fuse a protein to the PTD. Anyways the papers I used to come to this conclusion are below and it would be nice if someone can verify/refute my interpretation of them.

 

http://www.esi2.us.es/~ramonrd/english/ptd4_techinfo2.pdf

http://cmm.ucsd.edu/Lab_Pages/dowdy/tat_mta.pdf

 

Also should mention that those papers only talk about using TAT as a PTD however I'm just talking about the general case where any given PTD is used.

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I have not read the papers, but often it does not make much of a difference whether the start codon is introduced or not. Sometimes the additional methionine (especially the sulfur residue) may prove to be problematic, but usually one just has to clone in-frame. The UTR obviously has to be removed, otherwise you would introduce some random aa sequences.

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Seems obvious but don't forget to also remove the stop codon (from the gene of interest or the PTD, depending on which end of the protein you're attaching it to) otherwise you won't get a fusion protein. I almost made that mistake once because I am silly.

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I have not read the papers, but often it does not make much of a difference whether the start codon is introduced or not. Sometimes the additional methionine (especially the sulfur residue) may prove to be problematic, but usually one just has to clone in-frame. The UTR obviously has to be removed, otherwise you would introduce some random aa sequences.

 

Right right. For some reason I had confused methionine for a stop codon. I think that the paper was assuming that whatever gene was being cloned in either had a stop codon or a promoter in it's 5' UTR, so I think as long as it doesn't have either of those between the PTD and the gene of interest it should be fine (ie if you were to use only the ORF of a gene).

 

However I'm wondering if the choice of restriction enzymes used to clone in a gene could cause problems. You don't think that any extra base pairs in between the PTD and the cloned in gene would cause a problem if they are only there because the restriction site didn't immediately follow the PTD? In that second link there are a couple of plasmid maps. In the first map it shows the MCS immediately following the PTD, which is TAT in those maps, but I'm going to guess that you don't need to use BamHI as one of your restriction sites since in the second plasmid map they put a tag in between the PTD and MCS. I guess what I'm asking is whether or not base pairs in between the PTD and cloning site of a gene would prevent the two from fusing as a protein, assuming those particular base pairs aren't part of any UTR and don't code for a stop codon.

Edited by 1101
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The important bit is generally that the whole construct is in-frame. I.e. if you translate it, you should get the correct overall AA sequence. Small aberrations are often not avoidable, but especially in those linker regions they tend not to be crucial (though as usual with recombinant protein expression there may be exceptions). If the additional base pairs result in frame-shift, the whole thing won´t work, obviously.

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The important bit is generally that the whole construct is in-frame. I.e. if you translate it, you should get the correct overall AA sequence. Small aberrations are often not avoidable, but especially in those linker regions they tend not to be crucial (though as usual with recombinant protein expression there may be exceptions). If the additional base pairs result in frame-shift, the whole thing won´t work, obviously.

 

Good to know. I have another question, all that needs to be done to make a PTD fusion plasmid is to clone in the PTD sequence somewhere in the MCS and then clone in a gene of interest downstream of that (presumably the closer the better) right? I think it's pretty obvious that it should work if the cloning is done right but I just want to check.

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Normally you would want to clone the PTD in first and the create or clone in an MCS after that. That way the plasmid is more convenient to clone fragments into. Also you would want to avoid cutting out the PTD when using restriction enzymes. So often e.g. a PCR construct with the PTD is created that can be ligated into the original vector, often in a way that does not result in a restriction site (unless you want to be able to remove the PTD sequence, then it should be a enzyme not present in the downstream MCS).

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Normally you would want to clone the PTD in first and the create or clone in an MCS after that. That way the plasmid is more convenient to clone fragments into. Also you would want to avoid cutting out the PTD when using restriction enzymes. So often e.g. a PCR construct with the PTD is created that can be ligated into the original vector, often in a way that does not result in a restriction site (unless you want to be able to remove the PTD sequence, then it should be a enzyme not present in the downstream MCS).

 

Would the only problem with cloning in the PTD after the MCS (or rather cloning it somewhere in the MCS) be the loss of restriction sites (assuming you wanted to avoid removing the PTD)? It wouldn't affect the creation of a fusion protein?

Edited by 1101
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It is more a matter of convenience. You could clone it into the MCS, but then you are limited in how you can clone in the subsequent protein to create the fusion. But functionally, if you get the stuff in frame into the construct you should be fine.

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It is more a matter of convenience. You could clone it into the MCS, but then you are limited in how you can clone in the subsequent protein to create the fusion. But functionally, if you get the stuff in frame into the construct you should be fine.

 

Thank you very much for your help.

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