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GFP fusion protein not expressing!


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#1 biology_06er

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Posted 27 June 2012 - 02:17 PM

Hi there,

I tried to make a GFP fusion protein (my gene of interest was fused to the C terminal of GFP) but no expression occured...I tested the expression of the two proteins alone and individually they worked but not when fused together..I tried a linker between the two..still no luck...could it just be that the fusion protein is somewhat toxic to the cell (doesn't seem likely aye..especially if the 2 alones are ok)

cheers,
b_06er

#2 Hong Zhan

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Posted 27 June 2012 - 08:15 PM

Hi there,

I tried to make a GFP fusion protein (my gene of interest was fused to the C terminal of GFP) but no expression occured...I tested the expression of the two proteins alone and individually they worked but not when fused together..I tried a linker between the two..still no luck...could it just be that the fusion protein is somewhat toxic to the cell (doesn't seem likely aye..especially if the 2 alones are ok)

cheers,
b_06er


Hi, for E.coli? it's N-ter tagging GFP right? Make sure you your construction that GFP do not disrupt the ORF of the gene. some GFP virant is not suitable in extracellular expression. Try other tag, as myc or flag or HA...Good luck.

#3 Drake1982

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Posted 07 July 2012 - 06:37 AM

Hi,
I had some similar problem a while ago ... but I tried both, a N- and a C-terminal fusion of gfp to my gene. What I encountered was simply that GFP fused to the N terminal of my protein did not work; but fusing it to the C terminal worked perfectly. You'll definitely need a linker in-between, because otherwise the protein might not fold correctly. Furthermore, as soon as you see fluorescence you can be very sure, that you protein is also correct folded (since GFP can only be expressed when it's folded correctly). So when you fuse GFP to the N-terminal of your protein, you might see fluorescence, but you would not know, whether your protein will be correctly folded.
So try to fuse GFP to the C-terminal of your protein and use a qualified linker! ;)

Good luck!




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