Not aware of any studied in particular. The question is a very important one, for any tag used as it is not a physiological event. I would suggest trying a number of different tags of different sizes to rule out any TAG induced observations. Another thing I try to avoid is over-expression systems that would cause excessive levels of protein expression from strong promoters if trying to be as physiological as possible.

At the end of the day, we can only use the tools available.

Good Luck!

One of the things you might consider is to check the growth rate between untransformed cells, transform with plasmid with your gene of interest, transform with plasmid with GFP only, transform with plasmid with GFP+gene of interest.

That will give you the answer but it also means 4x the work.

Hi,

Sometimes the position of the GFP can affect the function of the protein. Usually, I generate two different fusion proteins juste in case (GFP in N-ter and C-ter). If your GFP-protein localises like the endogenous one it's likely that it is functional.

Remember that the only way to be sure your protein is functional is to rescue the phenotype induced by the endogenous depletion with your GFP.

Hi, I think you will need adequate 'spacer' between your protein and your tag protein so they are not interfering with each other. GFP-tag is extesnsively used as reporter mark, I never know any cases where it interferes with the main protein function. However, your question is indeed interesting and plausible.

I've done extensive tagging with GFP and yes it can interfere with function. To minimize interference, I take into consideration whether there are domains at either end of the protein, such as a C terminal tail-anchor domain which can be identified by looking at a hydropathy plot of your protein and avoid tagging at that end...then I always include a "spacer" typically a 10 alanine linker between the protein and the tag, and lastly if functionality is critical I must have a way of confirming functionality after tagging, i.e. growth defect recovery or some other test for function. If there is no known way to test functionality but I've considered the two other things above, I just be sure to test several clones rather than rely on just one to be sure the results all agree.

I agree with everything posted so far. I have tagged many proteins with these kind of tags. It absolutely can interfere with function and even stability. Some proteins work with N-terminal tags, others with C-terminal, and some not at all. Like Tammy said, it is very good to have a functional assay to test your GFP fusion proteins. When GFP interferes with function (or expression etc.) I next try to use a smaller tag, like 3X FLAG or MYC, and I've had good results with these.

The bottom line is you have to try with your specific protein to find out if its going to work, and as suggested before, try both N and C-terminal tags. Then check for expression and functionality (if that is important for you).

Good luck!

Val

It is not true that GFP doesn't affect proteins. Sometimes it does and sometimes it doesn't.

There are many different scenarios;

1) GFP sequence is complementary to regions of the gene. This may cause the mRNA to fold into secondary structures by base complementarity between the gene sequence and GFP sequence. This is not too likely since we do not produce these GFP proteins and therefore our genes aren't likely to contain this sequence either. But this is not true for introns, which can mutate relatively freely so some intronic sequence may be complementary to GFP sequence by chance. If the mRNA or it's precursors (with introns) folds into secondary structures due to GFP sequence then maybe the mRNA will fail to be spliced/processed, fail to be exported from the nucleus to the cytoplasm or interfere with translation. This may cause none/low levels of the protein to be expressed and therefore low signal of fluorescence.

2) Sometimes we are interested in where a protein is, in the cell. So we can tag it with GFP. But proteins which respond to different signals or which have a defined cellular location under different conditions are 'told' to go to these locations by sequences in the amino acids. GFP can interfere with that. If a protein is needed in the endoplasmic reticulum (ER) or needs to be exported; its mRNA can be translated by ribosomes which sit on the ER. Whilst the protein is being synthesized, it is being fed through the ER membrane at the same time. GFP can interfere in this process.

3) GFP can interfere with the function of the protein. Maybe by causing the protein to fold differently. Maybe the active site of the protein is at the same site which GFP is fused. This could block the active site so that other molecules cannot bind or interact with the protein causing it to lose its function. Maybe now it can't interact with other proteins or can't bind to DNA and turn up or down the expression of other genes.

see Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Jennetta W. Hammond,1 T. Lynne Blasius,1 Virupakshi Soppina,1 Dawen Cai,1,2 and Kristen J. Verhey1,2 J. Cell Biol. Vol. 189 No. 6 1013–1025