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Is it possible to attach any protein with GFP and express together?


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6 replies to this topic

#1 Inbox

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Posted 25 February 2014 - 07:10 AM

Hi,

   Is it possible to attach any protein with GFP and express together? Thanks.



#2 bob1

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Posted 25 February 2014 - 02:47 PM

In theory, yes, in practice - no. 



#3 perneseblue

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Posted 25 February 2014 - 06:24 PM

No. GFP can cause steric hindrance by physically interfering with the function of a protein.

There is a possibility that it will also cause folding problem in your protein.

 

However if you're expressing your protein in a eukaryote system, you can try using 2A self cleaving peptides (P2A or T2A) to link your gene and GFP.  The 2A peptide will cause ribosomal pausing before restarting. So you get 1 mRNA that produces your protein and the GFP protein. So the expression of your protein is correlated to GFP.


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Posted 25 February 2014 - 08:45 PM

Thanks Bob & perneseblue. I want to use prokaryotic expression system. Can I still use 2A self cleaving peptide for linking gene with GFP. Which vector can be suitable for this type of study? Is there chance that my protein cause GFP to sterically inhibit not allowing to show its fluorescence. Can we measure fluoresce of GFP?



#5 perneseblue

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Posted 27 February 2014 - 07:46 PM

To the best of my knowledge the 2A only works in Eukaryotes (plants, animal and insects).  The 2A peptides that I know of will not work in bacteria systems. So no, the 2A peptide will not work in bacteria. (unless found otherwise)

 

It is very unlikely that your protein will interfere with GFP. GFP is very good at folding itself and is pretty much self contained. If there is any interference, it is more likely GFP interfering with your protein.

 

The Standard operating procedure with GFP tagging of an unknown protein is to make an N-terminal and C-terminal fusion protein and see which works.

N-GFP-linker-Gene-C

N-Gene-linker-GFP-C

 

Why do you want to tag your protein with GFP in a bacterial system? Are you interested in localisation of your protein in bacteria?


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Posted 01 March 2014 - 12:21 AM

I am looking for if I can differentiate cellular, membrane and extracellular proteins by this. ( Though I have not done enough background work).

 

@ Bob:- Why do you say practice no? There are reports of such GFP-protein fusion in E.coli.



#7 bob1

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Posted 01 March 2014 - 01:20 AM

So the theory is that you can tag GFP to pretty much any protein and the GPF will fold (it does this itself, independent of the other protein), and that much is true - but it happens that sometimes tagging a protein with GFP will somehow prevent the non-GFP part of the protein to incorrectly fold or cause a steric hindrance, or interfere with a signalling site/localization signal, and hence the protein is non-functional...so making the tagging with GFP not practicable.






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