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Expression of a GFP-tagged protein


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#1 MKN

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Posted 18 August 2009 - 09:06 AM

Hi all,

I have problems expressing a eGFP-tagged protein. I made a construct of my gene of interest (GOI) with the pEGFP-N1 vector. My protein of interest alone is 22kDa, and the eGFP is fused at the C-terminal of the protein. The cloning is between SacI and SalI sites. A Kozak sequence has been inserted. I transfected the construct into a mammalian cell line with nucleofection (Amaxa). The control vector is the empty vector pEGFP-N1, which also expresses the eGFP, and thus can indicate the transfection efficiency. eGFP expression was detected by FACS. In the transfection with empty vector, more than 80% of the cells contain eGFP (24h timepoint), while in the transfection with the GOI-eGFP fusion, there were only 8% cells positive for eGFP. The same thing is observed under the fluorescent microscope. I haven't checked the mRNA level yet (which I will do now), but I expect to observe increase in mRNA by about 3-fold. I have this expectation because in the last trial I used a construct without a Kozak sequence, and got 3-fold mRNA increase (24h) but only 4% cells with GOI-eGFP fusion...
Does anyone have a clue on what to do next? Should I do Western blot to see if the fusion protein is there (and thus can determine that translation has taken place and the protein is not degraded, but that there is incorrect folding)? In case there is incorrect folding, what can I do to help the protein fold?
Thanks very much!! :)

#2 laurequillo

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Posted 27 October 2009 - 09:46 PM

You could try a N-terminal GFP. Sometimes the position of the tag affects the folding of the protein

Edited by laurequillo, 27 October 2009 - 09:47 PM.

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#3 Penguin

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Posted 29 October 2009 - 07:00 AM

Is your GOI toxic? Have you codon optimised? Have you sequenced your construct to ensure the cloning is in-frame? Which cell line are you using? 293s are the best to start to test expression constructs, if it doesn't express in 293s its less likely to express in your cell line of interest

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