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Tagging a protein with a nucelar export signal


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#1 KPDE

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Posted 05 February 2009 - 01:51 PM

Does anyone have experience adding a nuclear export signal to a protein. They're a short peptide sequence which should make designing the construct easy but the hydrophobicity of the peptide has me worried. If I stuck it on the C-terminus would that end up altering the structure of the protein? Anyone with suggestions of the best context to but the signal into or with success stories, I'd be happy to hear your experience.

Thanks.

#2 cellcounter

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Posted 05 February 2009 - 02:15 PM

Does anyone have experience adding a nuclear export signal to a protein. They're a short peptide sequence which should make designing the construct easy but the hydrophobicity of the peptide has me worried. If I stuck it on the C-terminus would that end up altering the structure of the protein? Anyone with suggestions of the best context to but the signal into or with success stories, I'd be happy to hear your experience.

Thanks.

I have no experience with tagging NES. But what choices do we have? N and C-terminus. I doubt anyone can predict which one would work better with your particular protein.

I suggest, if you don't mind putting some additional work, make both. N and C-terminus tagging sometimes have dramatic affect on protein strucutre, function or localization, so it would be a prudent thing to do, unless all you are looking for is pull-down experiments with his or flag-tags. And even there sometimes there are problems.

I also hope somebody can throw more light on this topic!

#3 KPDE

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Posted 05 February 2009 - 03:07 PM

Does anyone have experience adding a nuclear export signal to a protein. They're a short peptide sequence which should make designing the construct easy but the hydrophobicity of the peptide has me worried. If I stuck it on the C-terminus would that end up altering the structure of the protein? Anyone with suggestions of the best context to but the signal into or with success stories, I'd be happy to hear your experience.

Thanks.

I have no experience with tagging NES. But what choices do we have? N and C-terminus. I doubt anyone can predict which one would work better with your particular protein.

I suggest, if you don't mind putting some additional work, make both. N and C-terminus tagging sometimes have dramatic affect on protein strucutre, function or localization, so it would be a prudent thing to do, unless all you are looking for is pull-down experiments with his or flag-tags. And even there sometimes there are problems.

I also hope somebody can throw more light on this topic!


Tagging both ends would be the thing to do except that the gene is for a cell surface protein that may be cleaved before going to the nucleus. The thing I was curious about is if there was any value to putting it in an internal position and if there was some criteria for optimizing the context to put it in




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