3 replies to this topic

[yvette9995]

Hi everyone.
I'm at my wit's end trying to overexpress a gfp-tagged protein in human breast cancer cells.  I have a (purchased) plasmid with the ORF of my gene of interest.  I PCR-amplified this ORF and added a kozak sequence (GCCGCC) as well as appropriate flanking restriction sites.  I subcloned this into pEGFP-C1 and screened the resultant colonies.  Sequencing results show that  is in frame with pEGFP.  I transfected the cells-- and got nothing.  A transfection with empty vector worked just fine.  Any suggestions for troubleshooting? one thing that has me very confused is that my protein is downstream of egfp, so how is it possible that I am not getting a signal? Thanks!

Edited by yvette9995, 02 October 2009 - 01:17 PM.

[stardust]

Perhaps the EGFP and your protein are now too close together to fold properly. Did you place a linker between EGFP and your protein? If it is downstream of EGFP why the Kozak sequence? Did you remove the EGFP stop codon? Perform RT-PCR to see if your fgusion mRNA is expressed...

Stardust

[eldon]

stardust, on Oct 3 2009, 06:14 AM, said:

Perhaps the EGFP and your protein are now too close together to fold properly. Did you place a linker between EGFP and your protein? If it is downstream of EGFP why the Kozak sequence? Did you remove the EGFP stop codon? Perform RT-PCR to see if your fgusion mRNA is expressed...

Stardust

All excellent suggestions. Might want to try a western blot as well.

One more is, if all else fails and you in fact haven't accidently left the stop codon in place is to try the fusion with your gene of interest at the Nterm of eGFP. The fluorescent moiety of GFP is mostly Cterm if I recall correctly. Plus, it's a good control...nothing worse than the localization of your protein being different if GFP is fused Cterm vs Nterm.

[yvette9995]

thanks for the suggestions.  The reason the kozak sequence is there is because I originally subcloned the ORF into pcDNA3.1, which apparently may require it.  So it's there incidentally- is this a problem?  The sequence was subcloned into the MCS of pEGFP upstream of the stop sites, so that isn't the issue.  I've thought about the possibility of adding a linker, but I was under the impression that it's only usually necessary if you're worried about altering the activity of your protein.  I also have pmCherry-N1 with compatible restriction sites that I could subclone into to see if it works better as a C-terminal tag.  If I do have to add a linker, how do I determine which linker to use? Thanks again- I am in a lab with only one other grad student and few people around to talk to about this kind of thing.