I'm currently trying to design several dozen new GFP-linked fusion proteins for an upcoming project, but I struggle to find guidelines of how to design the linker between protein of interest and GFP. I'm aware that N- or C-terminal linkage can affect expression, targetting and functionality of the protein of interest and have established for my specific candidate proteins which terminus to use. However, I am uncertain of how long the linker should be and which amino acids to favour or to avoid. I would be very thankful if someone could point me into the right direction! I should mention that I have complete flexibility, as I'm going to synthesize the constructs from scratch.
Hi Sven, I guess there is no 100% certainty how a perfect linker should look like. It actually depends on many variables e.g. the structure of your protein of interest. You can also use secondary structure prediction tools to guide fusion protein design. If there are unstructured N- or C-terminal extensions you may not need a linker at all. If your protein of interest recruits binding partners via residues close to the N- or C-terminus I would rather not fuse GFP close to this site etc. You can actually use linkers to generate distance (e.g. PAPAP linkers) or you can allow for lots of flexibility (e.g. using GGGGSn linkers). Having said that if you are making GFP fusion proteins assuming the GFP is just a fluorescence tag that is not required to do something fancy apart from beeing green a 2 amino acid linker (resembling e.g. a restriction site between GFP and your protein of interst) or a GGGGS linker may be absolutely sufficient. There is some literature on linker that may be helpful : Article Fusion Protein Linkers: Property, Design and Functionality https://academic.oup.com/peds/article/27/10/325/1455209 https://academic.oup.com/peds/article/13/5/309/1505267 On more thing... make sure to delete the N-terminal methionine of GFP if you use it as a C-terminal fusion to prevent the expression of unfused GFP. Hope that helps, best of luck Jens
Dear Jens Mark Moll,
I have a question regarding your last piece of advice, in which you recommend deleting the N-terminal ATG of GFP to prevent the expression of unfused GFP. I have a construct with a receptor fused with mCherry (separated by a short linker), but mCherry contains the ATG codon at the N-term (and hence, the methionine). However, my receptor does not contain a stop codon at the C-terminal. Therefore, I should not expect that GFP is synthesized unfused, should I? Given that translation termination will not occur, I assumed the ORF would be translated all as a fusion protein, and simply the ATG would be translated to a methionine.
Thank you in advance for your help.
Patricia
Hi, Patricia Molina Molina, I think you are right. Even though the mCherry has ATG(hence, the methionine) following the c-terminal of the receptor(with no stop code), the cell will translate the whole ORF as one mRNA(One ATG and One stop code). The inside ATG in this ORF will not affect the expression of your fusion protein.
Bin
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