Hello everyone, I am preparing a fusion protein with mCherry and I have doubts about the type of linker to use. I have seen in the literature that sometimes flexible linkers of this type of sequence are used: (Gly-Gly-Gly-Gly-Ser)n. Can an expert in the field confirm whether this residue composition is the most suitable? What would be the optimal length?
Thank you for your help.
I agree with your feeling that Gly makes a good linker.
I think length is largely empirical to your specific protein and your example seems completely reasonable.
Some constructs will localize and some will not, you just need to test it out.
Hi there,
I have evaluated the effect of (Gly4Ser)2 spacer on mIL3-mediated diphteria toxin toxicity: the presence of the spacer significantly improved toxicity. If interested, the article published in FEBS Letters in 1997 is accessible from my researchgate contribution page.
Thank you all for your answers, I will try this linker first: Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly
And let's see how it goes!
Thanks!
Hi Ferdinand,
Yes, it worked. This is the linker that I finally used: Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly
I can observe good mCherry expression (C-terminal to the linker) by fluorescence microscopy. I still have to run some tests to validate that my protein of interest (N-terminal to the linker) is still present as a fusion protein (not cleaved). But even that's the case I assume it will be due to the sequence of my protein and not the linker.
here is a nice homepage for those interested:
http://parts.igem.org/Protein_domains/Linker
I will try the Gly-Gly-Ser-Gly linker.
cheers, Milan
Have you ever noticed that Gly-Ser linker is cleaved by proteases or that the ribosome falls off bevore the whole fusion protein is produced? I have problems with different lenghts of the produced proteins and I don't know why.
Hi Sasa,
In my case the entire protein is produced (using the Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly linker). The fusion protein has the predicted size, as determined by Western blot. No cleaving products are detected.
which linker is better for cleaving in tissue for protein vaccine?
(we want to link some peptide with suitable linker)
what linker you suggest ?
Hi Tahoora,
Do you mean a linker that gets cleaved upon tissue uptake? That's interesting but unfortunately I don't have experience in this topic, sorry. Perhaps you can read this review that I just found after a google search: https://www.ncbi.nlm.nih.gov/pubmed/21880494
I hope you can find good suggestions there!
Hi Tahoora,
Did you find any linkers suitable for your work? I am looking for similar one.
There are linkers based on the same principle than the (GGGGS)n which I find superior as they don't have repeats that can lead to homologous recombination. One example: GSAGSAAGSGEF.
ref for the GSAGSAAGSGEF liner : Waldo, G.S., Standish, B.M., Berendzen, J., and Terwilliger, T.C. (1999). Rapid protein-folding assay using green fluorescent protein. Nat. Biotechnol. 17, 691–695.
ref for other similar linkers: Chen, X., Zaro, J.L., and Shen, W.-C. (2013). Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369.
You may not use redundant linker, just direct link sometime better than any link
Hi Anchel,
I know that I am a little bit in late.
I am really curious to know if your protein with the mCherry tag at the 3-terminal was still functional. If I can ask, which protein it was?
Thank you very much
Hi Suman,
In my study the functionality of the protein was not relevant, so we didn't test it.
If you suspect the C-terminal in your protein is part of a domain important for its function perhaps you can make an additional construct with the tag in a different position. But keep in mind that placing the tag N-terminal will very likely change the normal intracellular distribution since localization sequences are usually there.
Sometimes, tagging a protein without changing its localization and function could be very challenging, perhaps you will have to test multiple approaches. Or tag it internally, by click-chemistry for example?
Maybe others have better suggestions?
Hi.
(Pro-Gly-Pro-Gly) linker could be a good choice for fusion proteins and subunite vaccine designing. it worked on my work:
Hi anchel
The (pro-gly-pro-gly)n linker could be a good choice for fusion proteins specially subunit vaccines.
It was tested here experimentally: Article Constructing and transient expression of a gene cassette con...
And bioinformatically analysis: Article Bioinformatic analysis of different fusions of ipaD, PA20 an...
Hope to succeed.
Amy Johnson
, I do not think the "ATG needs to occur only once in a fusion gene" make much sense. Take for example the NhaA gene in E. coli (my PhD subjetc :-), available here https://www.ncbi.nlm.nih.gov/nuccore/NC_002695.1?report=fasta&from=16157&to=17323As you can see there are multiple Methionines in the protein sequence, and since the only codon for Met is ATG there are multiple ATGs... Interestingly the coding region start with CTC, not ATG...
Yoram
This might be a bit extra, but you might be able to just create a recombinant gene with a linker sequence containing a blend of both genes (assuming all subunits are helical) by finding the nearest flanking restriction site (3’ for upstream and 5’ for the downstream gene by looking up the sequence on BLAST, and selecting a source organism with both restriction sites enclosing filler DNA - it’s tricky to articulate what I mean, but it’s basically crossing your fingers and praying that you end up with the three sequences ligating together. (I’ve never done this before, I’m only a freshman in college, but I’ve been computing the source organisms I’d need to make a botulinum toxin detector protein for mandatory quality-control checks for about 2-ish years now without any expert input, so I could be 100% wrong, since it’s literally a very chemically involved game of “find the very short oligonucleotide in the haystack.”
Amy Johnson
, I don't know if I fully understand what you want to do but, if you are wondering if a second ATG is necessary to express the second gene as a fusion protein, the answer is no. If you don't include it, the first Met of GFP will not be in the sequence but, as long as the entire open reading frame is maintained, the cassette will be expressed using only the TSS of the gene at 5'. The ribosomes will scan the transcript until they find the first AUG, from where protein synthesis will begin and continue all the way though the linker and GFP (subsequent AUGs will be translated as Met, but not used as start codons). In fact, for the design to work as a fusion protein, you need to remove GFP's 5'-UTR or else the ribosome may find a premature stop codon and drop off before continuing with GFP, or it will be expressed separately (not as fusion protein).I hope this helps and my apologies in advance if I missed the point of your question.
XTEN linker is anther flexible linker and found very good in Cas9 linked deaminase. It may be a good choice for nuclease-like enzymes. See below an example:
Article Engineering of high-precision base editors for site-specific...
Another interesting paper on linkers:
Article IFLinkC: An iterative functional linker cloning strategy for...
- Badges
- Science method